Apparatus for polymer synthesis

ABSTRACT

A method and apparatus for preparation of a substrate containing a plurality of sequences. Photoremovable groups are attached to a surface of a substrate. Selected regions of the substrate are exposed to light so as to activate the selected areas. A monomer, also containing a photoremovable group, is provided to the substrate to bind at the selected areas. The process is repeated using a variety of monomers such as amino acids until sequences of a desired length are obtained. Detection methods and apparatus are also disclosed.

COPYRIGHT NOTICE

[0001] A portion of the disclosure of this patent document containsmaterial which is subject to copyright protection. The copyright ownerhas no objection to the facsimile reproduction by anyone of the patentdocument or the patent disclosure as it appears in the Patent andTrademark Office patent file or records, but otherwise reserves allcopyright rights whatsoever.

BACKGROUND OF THE INVENTION

[0002] The present inventions relate to the synthesis and placementmaterials at known locations. In particular, one embodiment of theinventions provides a method and associated apparatus for preparingdiverse chemical sequences at known locations on a single substratesurface. The inventions may be applied, for example, in the field ofpreparation of oligomer, peptide, nucleic acid, oligosaccharide,phospholipid, polymer, or drug congener preparation, especially tocreate sources of chemical diversity for use in screening for biologicalactivity.

[0003] The relationship between structure and activity of molecules is afundamental issue in the study of biological logical systemsStructure-activity relationships are important in understanding, forexample, the function of enzymes, the ways in which cells communicatewith each other, as well as cellular control and feedback systems.

[0004] Certain macromolecules are known to interact and bind to othermolecules having a very specific three-dimensional spatial andelectronic distribution. Any large molecule having such specificity canbe considered a receptor, whether it is an enzyme catalyzing hydrolysisof a metabolic intermediate, a cell-surface protein mediating membranetransport of ions, a glycoprotein serving to identify a particular cellto its neighbors, an IgG-class antibody circulating in the plasma, anoligonucleotide sequence of DNA in the nucleus, or the like. The variousmolecules which receptors selectively bind are known as ligands.

[0005] Many assays are available for measuring the binding affinity ofknown receptors and ligands, but the information which can be gainedfrom such experiments is often limited by the number and type of ligandswhich are available. Novel ligands are sometimes discovered by chance orby application of new techniques for the elucidation of molecularstructure, including x-ray crystallographic analysis and recombinantgenetic techniques for proteins.

[0006] Small peptides are an exemplary system for exploring therelationship between structure and function in biology. A peptide is asequence of amino acids. When the twenty naturally occurring amino acidsare condensed into polymeric molecules they form a wide variety ofthree-dimensional configurations, each resulting from a particular aminoacid sequence and solvent condition. The number of possiblepentapeptides of the 20 naturally occurring amino acids, for example, is20⁵ or 3.2 million different peptides. The likelihood that molecules ofthis size might be useful in receptor-binding studies is supported byepitope analysis studies showing that some antibodies recognizesequences as short as a few amino acids with high specificity.Furthermore, the average molecular weight of amino acids puts smallpeptides in the size range of many currently useful pharmaceuticalproducts.

[0007] Pharmaceutical drug discovery is one type of research whichrelies on such a study of structure-activity relationships. In mostcases, contemporary pharmaceutical research can be described as theprocess of discovering novel ligands with desirable patterns ofspecificity for biologically important receptors. Another example isresearch to discover new compounds for use in agriculture, such aspesticides and herbicides.

[0008] Sometimes, the solution to a rational process of designingligands is difficult or unyielding. Prior methods of preparing largenumbers of different polymers have been painstakingly slow when used ata scale sufficient to permit effective rational or random screening. Forexample, the “Merrifield” method (J. Am. Chem. Soc. (1963) 85:2149-2154,which is incorporated herein by reference for all purposes) has beenused to synthesize peptides on a solid support. In the Merrifieldmethod, an amino acid is covalently bonded to a support made of aninsoluble polymer. Another amino acid with an alpha protected group isreacted with the covalently bonded amino acid to form a dipeptide. Afterwashing, the protective group is removed and a third amino acid with analpha protective group is added to the dipeptide. This process iscontinued until a peptide of a desired length and sequence is obtained.Using the Merrifield method, it is not economically practical tosynthesize more than a handful of peptide sequences in a day.

[0009] To synthesize larger numbers of polymer sequences, it has alsobeen proposed to use a series of reaction vessels for polymer synthesis.For example, a tubular reactor system may be used to synthesize a linearpolymer on a solid phase support by automated sequential addition ofreagents. This method still does not enable the synthesis of asufficiently large number of polymer sequences for effective economicalscreening.

[0010] Methods of preparing a plurality of polymer sequences are alsoknown in which a foraminous container encloses a known quantity ofreactive particles, the particles being larger in size than foramina ofthe container. The containers may be selectively reacted with desiredmaterials to synthesize desired sequences of product molecules. As withother methods known in the art, this method cannot practically be usedto synthesize a sufficient variety of polypeptides for effectivescreening.

[0011] Other techniques have also been described. These methods includethe synthesis of peptides on 96 plastic pins which fit the format ofstandard microtiter plates. Unfortunately, while these techniques havebeen somewhat useful, substantial problems remain. For example, thesemethods continue to be limited in the diversity of sequences which canbe economically synthesized and screened.

[0012] From the above, it is seen that an improved method and apparatusfor synthesizing a variety of chemical sequences at known locations isdesired.

SUMMARY OF THE INVENTION

[0013] An improved method and apparatus for the preparation of a varietyof polymers is disclosed.

[0014] In one preferred embodiment, linker molecules are provided on asubstrate. A terminal end of the linker molecules is provided with areactive functional group protected with a photoremovable protectivegroup. Using lithographic methods, the photoremovable protective groupis exposed to light and removed from the linker molecules in firstselected regions. The substrate is then washed or otherwise contactedwith a first monomer that reacts with exposed functional groups on thelinker molecules. In a preferred embodiment, the monomer is an aminoacid containing a photoremovable protective group at its amino orcarboxy terminus and the linker molecule terminates in an amino orcarboxy acid group bearing a photoremovable protective group.

[0015] A second set of selected regions is, thereafter, exposed to lightand the photoremovable protective group on the linker molecule/protectedamino acid is removed at the second set of regions. The substrate isthen contacted with a second monomer containing a photoremovableprotective group for reaction with exposed functional groups. Thisprocess is repeated to selectively apply monomers until polymers of adesired length and desired chemical sequence are obtained. Photolabilegroups are then optionally removed and the sequence is, thereafter,optionally capped. Side chain protective groups, if present, are alsoremoved.

[0016] By using the lithographic techniques disclosed herein, it ispossible to direct light to relatively small and precisely knownlocations on the substrate. It is, therefore, possible to synthesizepolymers of a known chemical sequence at known locations on thesubstrate.

[0017] The resulting substrate will have a variety of uses including,for example, screening large numbers of polymers for biologicalactivity. To screen for biological activity, the substrate is exposed toone or more receptors such as antibody whole cells, receptors onvesicles, lipids, or any one of a variety of other receptors. Thereceptors are preferably labeled with, for example, a fluorescentmarker, radioactive marker, or a labeled antibody reactive with thereceptor. The location of the marker on the substrate is detected with,for example, photon detection or autoradiographic techniques. Throughknowledge of the sequence of the material at the location where bindingis detected, it is possible to quickly determine which sequence bindswith the receptor and, therefore, the technique can be used to screenlarge numbers of peptides. Other possible applications of the inventionsherein include diagnostics in which various antibodies for particularreceptors would be placed on a substrate and, for example, blood serawould be screened for immune deficiencies. Still further applicationsinclude, for example, selective “doping” of organic materials insemiconductor devices, and the like.

[0018] In connection with one aspect of the invention an improvedreactor system for synthesizing polymers is also disclosed. The reactorsystem includes a substrate mount which engages a substrate around aperiphery thereof. The substrate mount provides for a reactor spacebetween the substrate and the mount through or into which reactionfluids are pumped or flowed. A mask is placed on or focused on thesubstrate and illuminated so as to deprotect selected regions of thesubstrate in the reactor space. A monomer is pumped through the reactorspace or otherwise contacted with the substrate and reacts with thedeprotected regions. By selectively deprotecting regions on thesubstrate and flowing predetermined monomers through the reactor space,desired polymers at known locations may be synthesized.

[0019] Improved detection apparatus and methods are also disclosed. Thedetection method and apparatus utilize a substrate having a largevariety of polymer sequences at known locations on a surface thereof.The substrate is exposed to a fluorescently labeled receptor which bindsto one or more of the polymer sequences. The substrate is placed in amicroscope detection apparatus for identification of locations wherebinding takes place. The microscope detection apparatus includes amonochromatic or polychromatic light source for directing light at thesubstrate, weans for detecting fluoresced light from the substrate, andmeans for determining a location of the fluoresced light. The means fordetecting light fluoresced on the substrate may in some embodimentsinclude a photon counter. The means for determining a location of thefluoresced light may include an x/y translation table for the substrate.Translation of the slide and data collection are recorded and managed byan appropriately programmed digital computer.

[0020] A further understanding of the nature and advantages of theinventions herein may be realized by reference to the remaining portionsof the specification and the attached drawings.

BRIEF DESCRIPTION OF THE FIGURES

[0021]FIG. 1 illustrates masking and irradiation of a substrate at afirst location. The substrate is shown in cross-section;

[0022]FIG. 2 illustrates the substrate after application of a monomer“A”;

[0023]FIG. 3 illustrates irradiation of the substrate at a secondlocation;

[0024]FIG. 4 illustrates the substrate after application of monomer “B”;

[0025]FIG. 5 illustrates irradiation of the “A” monomer;

[0026]FIG. 6 illustrates the substrate after a second application of“B”,

[0027]FIG. 7 illustrates a completed substrate;

[0028]FIGS. 8A and 8B illustrate alternative embodiments of a reactorsystem for forming a plurality of polymers on a substrate;

[0029]FIG. 9 illustrates a detection apparatus for locating fluorescentmarkers on the substrate;

[0030] FIGS. 10A-10B illustrate the method as it is applied to theproduction of the trimers of monomers “A” and “B”;

[0031]FIGS. 11A, 11B, AND 11C are fluorescence traces for standardfluorescent beads;

[0032]FIGS. 12A and 12B are fluorescence curves for NVOC slides notexposed and exposed to light respectively;

[0033]FIGS. 13A to 13D are fluorescence plots of slides exposed through100 μm, 50 μm, 20 μm, and 10 μm masks;

[0034]FIG. 14 illustrates fluorescence of a slide with the peptide YGGFLon selected regions of its surface which has been exposed to labeledHerz antibody specific for this sequence;

[0035]FIGS. 15A to 15D illustrate formation of and a fluorescence plotof a slide with a checkerboard pattern of YGGFL and GGFL exposed tolabeled Herz antibody. FIG. 15C illustrates a 500×500 μm mask which hasbeen focused on the substrate according to FIG. 8A while FIG. 15Dillustrates a 50×50 μm mask placed in direct contact with the substratein accord with FIG. 8B;

[0036]FIG. 16 is a fluorescence plot of YGGFL and PGGFL synthesized in a50 μm checkerboard pattern;

[0037]FIG. 17 is a fluorescence plot of YPGGFL and YGGFL synthesized ina 50 μm checkerboard pattern;

[0038]FIGS. 18A and 18B illustrate the mapping of sixteen sequencessynthesized on two different glass slides;

[0039]FIG. 19 is a fluorescence plot of the slide illustrated in FIG.18A; and

[0040]FIG. 20 is a fluorescence plot of the slide illustrated in FIG.10B.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS CONTENTS

[0041] I. Glossary

[0042] II. General

[0043] III. Polymer Synthesis

[0044] IV. Details of One Embodiment of a Reactor System

[0045] V. Details of one Embodiment of a Fluorescent Detection Device

[0046] VI. Determination of Relative Binding Strength of Receptors

CONTENTS (Cont'd)

[0047] VII. Examples

[0048] A. Slide Preparation

[0049] B. Synthesis of Eight Trimers of “A” and “B”

[0050] C. Synthesis of a Dimer of an Aminopropyl Group and a FluorescentGroup

[0051] D. Demonstration of Signal Capability

[0052] E. Determination of the Number of Molecules Per Unit Area

[0053] F. Removal of NVOC and Attachment of a Fluorescent Marker

[0054] G. Use of a Mask in Removal of NVOC

[0055] H. Attachment of YGGFL and Subsequent Exposure to Herz Antibodyand Goat Antimouse

[0056] I. Monomer-by-Monomer Formation of YGGFL and Subsequent Exposureto Labeled Antibody

[0057] J. Monomer-by-Monomer Synthesis of YGGFL and PGGFL

[0058] K. Monomer-by Monomer Synthesis of YGGFL and YPGGFL

[0059] L. Synthesis of an Array of Sixteen Different Amino AcidSequences and Estimation of Relative Binding Affinity to Herz Antibody

[0060] VIII. Illustrative Alternative Embodiment

[0061] IX. Conclusion

[0062] I. Glossary

[0063] The following terms are intended to have the following generalmeanings as they are used herein:

[0064] 1. Complementary: Refers to the topological compatibility ormatching together of interacting surfaces of a ligand molecule and itsreceptor. Thus, the receptor and its ligand can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other.

[0065] 2. Epitope: The portion of an antigen molecule which isdelineated by the area of interaction with the subclass of receptorsknown as antibodies.

[0066] 3. Ligand: A ligand is a molecule that is recognized by aparticular receptor. Examples of ligands that can be investigated bythis invention include, but are not restricted to, agonists andantagonists for cell membrane receptors, toxins and venoms, viralepitopes, hormones (e.g., opiates, steroids, etc.), hormone receptors,peptides, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars,oligonucleotides, nucleic acids, oligosaccharides, proteins, andmonoclonal antibodies.

[0067] 4. Monomer: A member of the set of small molecules which can bejoined together to form a polymer. The set of monomers includes but isnot restricted to, for example, the set of common L-amino acids, the setof D-amino acids, the set of synthetic amino acids, the set ofnucleotides and the set of pentoses and hexoses. As used herein,monomers refers to any member of a basis set for synthesis of a polymer.For example, dimers of L-amino acids form a basis set of 400 monomersfor synthesis of polypeptides. Different basis sets of monomers may beused at successive steps in the synthesis of a polymer.

[0068] 5. Peptide: A polymer in which the monomers are alpha amino acidsand which are joined together through amide bonds and alternativelyreferred to as a polypeptide. In the context of this specification itshould be appreciated that the amino acids may be the L-optical isomeror the D-optical isomer. Peptides are more than two amino acid monomerslong, and often more than 20 amino acid monomers long. Standardabbreviations for amino acids are used (e.g., P for proline). Theseabbreviations are included in Stryer, Biochemstry, Third Ed., 1988,which is incorporated herein by reference for all purposes.

[0069] 6. Radiation: Energy which may be selectively applied includingenergy having a wavelength of between 10⁻¹⁴ and 10⁴ meters including,for example, electron beam radiation, gamma radiation, x-ray radiation,ultra-violet radiation, visible light, infrared radiation, microwaveradiation, and radio waves. “Irradiation” refers to the application ofradiation to a surface.

[0070] 7. Receptor: A molecule that has an affinity for a given ligand.Receptors may be naturally-occuring or manmade molecules. Also, they canbe employed in their unaltered state or as aggregates with otherspecies. Receptors may be attached, covalently or noncovalently, to abinding member, either directly or via a specific binding substance.Examples of receptors which can be employed by this invention include,but are not restricted to, antibodies, cell membrane receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells or other materials), drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles. Receptorsare sometimes referred to in the art as anti-ligands. As the termreceptors is used herein, no difference in meaning is intended. A“Ligand Receptor Pair” is formed when two macromolecules have combinedthrough molecular recognition to form a complex.

[0071] Other examples of receptors which can be investigated by thisinvention include but are not restricted to:

[0072] a) Microorganism receptors: Determination of ligands which bindto receptors, such as specific transport proteins or enzymes essentialto survival of microorganisms, is useful in a new class of antibiotics.Of particular value would be antibiotics against opportunistic fungi,protozoa, and those bacteria resistant to the antibiotics in currentuse.

[0073] b) Enzymes: For instance, the binding site of enzymes such as theenzymes responsible for cleaving neurotransmitters; determination ofligands which bind to certain receptors to modulate the action of theenzymes which cleave the different neurotransmitters is useful in thedevelopment of drugs which can be used in the treatment of disorders ofneurotransmission.

[0074] c) Antibodies: For instance, the invention may be useful ininvestigating the ligand-binding site on the antibody molecule whichcombines with the epitope of an antigen of interest; determining asequence that mimics an antigenic epitope may lead to the development ofvaccines of which the immunogen is based on one or more of suchsequences or lead to the development of related diagnostic agents orcompounds useful in therapeutic treatments such as for autoimmunediseases (e.g., by blocking the binding of the “self” antibodies).

[0075] d) Nucleic Acids: Sequences of nucleic acids may be synthesizedto establish DNA or RNA binding sequences.

[0076] e) Catalytic Polypeptides: Polymers, preferably polypeptides,which are capable of promoting a chemical reaction involving theconversion of one or more reactants to one or more products. Suchpolypeptides generally include a binding site specific for at least onereactant or reaction intermediate and an active functionality proximateto the binding site, which functionality is capable of chemicallymodifying the bound reactant. Catalytic polypeptides are described in,for example, U.S. application Ser. No. 404,920, which is incorporatedherein by reference for all purposes.

[0077] f) Hormone receptors: For instance, the receptors for insulin andgrowth hormone. Determination of the ligands which bind with highaffinity to a receptor is useful in the development of, for example, anoral replacement of the daily injections which diabetics must take torelieve the symptoms of diabetes, and in the other case, a replacementfor the scarce human growth hormone which can only be obtained fromcadavers or by recombinant DNA technology. Other examples are thevasoconstrictive hormone receptors; determination of those ligands whichbind to a receptor may lead to the development of drugs to control bloodpressure.

[0078] g) Opiate receptors: Determination of ligands which bind to theopiate receptors in the brain is useful in the development ofless-addictive replacements for morphine and related drugs.

[0079] 8. Substrate: A material having a rigid or semi-rigid surface. Inmany embodiments, at least one surface of the substrate will besubstantially flat, although in some embodiments it may be desirable tophysically separate synthesis regions for different polymers with, forexample, wells, raised regions, etched trenches, or the like. Accordingto other embodiments, small beads may be provided on the surface whichmay be released upon completion of the synthesis.

[0080] 9. Protective Group: A material which is bound to a monomer unitand which may be spatially removed upon selective exposure to anactivator such as electromagnetic radiation. Examples of protectivegroups with utility herein include Nitroveratryloxy carbonyl,Nitrobenzyloxy carbonyl, Dimethyl dimethoxybenzyloxy carbonyl,5-Bromo-7-nitroindolinyl, o-Hydroxy-α-methyl cinnamoyl, and2-Oxymethylene anthraquinone. Other examples of activators include ionbeams, electric fields magnetic fields, electron beams, x-ray, and thelike.

[0081] 10. Predefined Region: A predefined region is a localized area ona surface which is, was, or is intended to be activated for formation ofa polymer. The predefined region may have any convenient shape, e.g.,circular, rectangular, elliptical, wedge-shaped, etc. For the sake ofbrevity herein, “predefined regions” are sometimes referred to simply as“regions.”

[0082] 11. Substantially Pure: A polymer is considered to be“substantially pure” within a predefined region of a substrate when itexhibits characteristics that distinguish it from other predefinedregions. Typically, purity will be measured in terms of biologicalactivity or function as a result of uniform sequence. Suchcharacteristics will typically be measured by way of binding with aselected ligand or receptor.

[0083] II. General

[0084] The present invention provides methods and apparatus for thepreparation and use of a substrate having a plurality of polymersequences in predefined regions. The invention is described hereinprimarily with regard to the preparation of molecules containingsequences of amino acids, but could readily be applied in thepreparation of other polymers. Such polymers include, for example, bothlinear and cyclic polymers of nucleic acids, polysaccharides,phospholipids, and peptides having either α-, β-, or ω-amino acids,heteropolymers in which a known drug is covalently bound to any of theabove, polyurethanes, polyesters, polycarbonates, polyureas, polyamides,polyethyleneimines, polyarylene sulfides. polysiloxanes, polyimides,polyacetates, or other polymers which will be apparent upon review ofthis disclosure. In a preferred embodiment, the invention herein is usedin the synthesis of peptides.

[0085] The prepared substrate may, for example, be used in screening avariety of polymers as ligands for binding with a receptor, although itwill be apparent that the invention could be used for the synthesis of areceptor for binding with a ligand. The substrate disclosed herein willhave a wide variety of other uses. Merely by way of example, theinvention herein can be used in determining peptide and nucleic acidsequences which bind to proteins, finding sequence-specific bindingdrugs, identifying epitopes recognized by antibodies, and evaluation ofa variety of drugs for clinical and diagnostic applications, as well ascombinations of the above.

[0086] The invention preferably provides for the use of a substrate “S”with a surface. Linker molecules “L” are optionally provided on asurface of the substrate. The purpose of the linker molecules, in someembodiments, is to facilitate receptor recognition of the synthesizedpolymers.

[0087] Optionally, the linker molecules may be chemically protected forstorage purposes. A chemical storage protective group such as t-BOC(t-butoxycarbonyl) may be used in some embodiments. Such chemicalprotective groups would be chemically removed upon exposure to, forexample, acidic solution and would serve to protect the surface duringstorage and be removed prior to polymer preparation.

[0088] On the substrate or a distal end of the linker molecules, afunctional group with a protective group P₀ is provided. The protectivegroup P₀ may be removed upon exposure to radiation, electric fields,electric currents, or other activators to expose the functional group.

[0089] In a preferred embodiment, the radiation is ultraviolet (UV),infrared (IR), or visible light. As more fully described below, theprotective group may alternatively be an electrochemically-sensitivegroup which may be removed in the presence of an electric field. Instill further alternative embodiments, ion beams, electron beams, or thelike may be used for deprotection.

[0090] In some embodiments, the exposed regions and, therefore, the areaupon which each distinct polymer sequence is synthesized are smallerthan about 1 cm² or less than 1 mm². In preferred embodiments theexposed area is less than about 10,000 μm² or, more preferably, lessthan 100 μm² and may, in some embodiments, encompass the binding sitefor as few as a single molecule. Within these regions, each polymer ispreferably synthesized in a substantially pure form.

[0091] Concurrently or after exposure of a known region of the substrateto light, the surface is contacted with a first monomer unit M₁ whichreacts with the functional group which has been exposed by thedeprotection step. The first monomer includes a protective group P₁. P₁may or may not be the same as P₀.

[0092] Accordingly, after a first cycle, known first regions of thesurface may comprise the sequence:

S-L-M₁-P₁

[0093] while remaining regions of the surface comprise the sequence:

S-L-P₀.

[0094] Thereafter, second regions of the surface (which may include thefirst region) are exposed to light and contacted with a second monomerM₂ (which may or may not be the same as M₁) having a protective groupP2. P2 may or may not be the same as P₀ and P₁. After this second cycle,different regions of the substrate may comprise one or more of thefollowing sequences:

S-L-M₁-M₂-P₂

S-L-M₂-P₂

S-L-M₁-P₁ and/or

S-L-P₀.

[0095] The above process is repeated until the substrate includesdesired polymers of desired lengths. By controlling the locations of thesubstrate exposed to light and the reagents exposed to the substratefollowing exposure, the location of each sequence will be known.

[0096] Thereafter, the protective groups are removed from some or all ofthe substrate and the sequences are, optionally, capped with a cappingunit C. The process results in a substrate having a surface with aplurality of polymers of the following general formula:

S-[L]-(M_(i))-(M_(j))-(M_(k)) . . . (M_(x))-[C]

[0097] where square brackets indicate optional groups, and M_(i). . .M_(x) indicates any sequence of monomers. The number of monomers couldcover a wide variety of values, but in a preferred embodiment they willrange from 2 to 100.

[0098] In some embodiments a plurality of locations on the substratepolymers are to contain a common monomer subsequence. For example, itmay be desired to synthesize a sequence S-M₁-M₂-M₃ at first locationsand a sequence S-M₄-M₂-M₃ at second locations. The process wouldcommence with irradiation of the first locations followed by contactingwith M₁-P, resulting in the sequence S-M₁-P at the first location. Thesecond locations would then be irradiated and contacted with M₄-P,resulting in the sequence S-M₄-P at the second locations. Thereafterboth the first and second locations would be irradiated and contactedwith the dimer M₂-M₃, resulting in the sequence S-M₁-M₂-M₃ at the firstlocations and S-M₄-M₂-M₃ at the second locations. Of course, commonsubsequences of any length could be utilized including those in a rangeof 2 or more monomers, 2 to 100 monomers, 2 to 20 monomers, and a mostpreferred range of 2 to 3 monomers.

[0099] According to other embodiments, a set of masks is used for thefirst monomer layer and, thereafter, varied light wavelengths are usedfor selective deprotection. For example, in the process discussed above,first regions are first exposed through a mask and reacted with a firstmonomer having a first protective group P1, which is removable uponexposure to a first wavelength of light (e.g., IR). Second regions aremasked and reacted with a second monomer having a second protectivegroup P2, which is removable upon exposure to a second wavelength oflight (e.g., UV). Thereafter, masks become unnecessary in the synthesisbecause the entire substrate may be exposed alternatively to the firstand second wavelengths of light in the deprotection cycle.

[0100] The polymers prepared on a substrate according to the abovemethods will have a variety of uses including, for example, screeningfor biological activity. In such screening activities, the substratecontaining the sequences is exposed to an unlabeled or labeled receptorsuch as an antibody, receptor on a cell, phospholipid vesicle, or anyone of a variety of other receptors. In one preferred embodiment thepolymers are exposed to a first, unlabeled receptor of interest and,thereafter, exposed to a labeled receptor-specific recognition element,which is, for example, an antibody. This process will provide signalamplification in the detection stage.

[0101] The receptor molecules may bind with one or more polymers on thesubstrate. The presence of the labeled receptor and, therefore, thepresence of a sequence which binds with the receptor is detected in apreferred embodiment through the use of autoradiography, detection offluorescence with a charge-coupled device, fluorescence microscopy, orthe like. The sequence of the polymer at the locations where thereceptor binding is detected may be used to determine all or part of asequence which is complementary to the receptor.

[0102] Use of the invention herein is illustrated primarily withreference to screening for biological activity. The invention will,however, find many other uses. For example, the invention may be used ininformation storage (e.g., on optical disks), production of molecularelectronic devices, production of stationary phases in separationsciences, production of dyes and brightening agents, photography, and inimmobilization of cells, proteins, lectins, nucleic acids,polysaccharides and the like in patterns on a surface via molecularrecognition of specific polymer sequences. By synthesizing the samecompound in adjacent, progressively differing concentrations, a gradientwill be established to control chemotaxis or to develop diagnosticdipsticks which, for example, titrate an antibody against an increasingamount of antigen. By synthesizing several catalyst molecules in closeproximity, more efficient multistep conversions may be achieved by“coordinate immobilization.” Coordinate immobilization also may be usedfor electron transfer systems, as well as to provide both structuralintegrity and other desirable properties to materials such aslubrication, wetting, etc.

[0103] According to alternative embodiments, molecular biodistributionor pharmacokinetic properties may be examined. For example, to assessresistance to intestinal or serum proteases, polymers may be capped witha fluorescent tag and exposed to biological fluids of interest.

[0104] III. Polymer Synthesis

[0105]FIG. 1 illustrates one embodiment of the invention disclosedherein in which a substrate 2 is shown in cross-section. Essentially,any conceivable substrate may be employed in the invention. Thesubstrate may be biological, nonbiological, organic, inorganic, or acombination of any of these, existing as particles, strands,precipitates, gels, sheets, tubing, spheres, containers, capillaries,pads, slices, films, plates, slides, etc. The substrate may have anyconvenient shape, such as a disc, square, sphere, circle, etc. Thesubstrate is preferably flat but may take on a variety of alternativesurface configurations. For example, the substrate may contain raised ordepressed regions on which the synthesis takes place. The substrate andits surface preferably form a rigid support on which to carry out thereactions described herein. The substrate and its surface is also chosento provide appropriate light-absorbing characteristics. For instance,the substrate may be a polymerized Langmuir Blodgett film,functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon,or any one of a wide variety of gels or polymers such as(poly)tetrafluoro-ethylene, ethylene, (poly)vinylidenedifluoride,polystyrene, polycarbonate, or combinations thereof. Other substratematerials will be readily apparent to those of skill in the art uponreview of this disclosure. In a preferred embodiment the substrate isflat glass or single-crystal silicon with surface relief features ofless than 10 Å.

[0106] According to some embodiments, the surface of the substrate isetched using well known techniques to provide for desired surfacefeatures. For example, by way of the formation of trenches, v-grooves,mesa structures, or the like, the synthesis regions may be more closelyplaced within the focus point of impinging light, be provided withreflective “mirror” structures for maximization of light collection fromfluorescent sources, or the like.

[0107] Surfaces on the solid substrate will usually, though not always,be composed of the same material as the substrate. Thus, the surface maybe composed of any of a wide variety of materials, for example,polymers, plastics, resins, polysaccharides, silica or silica-basedmaterials, carbon, metals, inorganic glasses, membranes, or any of theabove-listed substrate materials. In some embodiments the surface mayprovide for the use of caged binding members which are attached firmlyto the surface of the substrate in accord with the teaching of copendingapplication Ser. No. 404,920, previously incorporated herein byreference. Preferably, the surface will contain reactive groups, whichcould be carboxyl, amino, hydroxyl, or the like. Most preferably, thesurface will be optically transparent and will have surface Si—OHfunctionalities, such as are found on silica surfaces.

[0108] The surface 4 of the substrate is preferably provided with alayer of linker molecules 6, although it will be understood that thelinker molecules are not required elements of the invention. The linkermolecules are preferably of sufficient length to permit polymers in acompleted substrate to interact freely with molecules exposed to thesubstrate. The linker molecules should be 6-50 atoms long to providesufficient exposure. The linker molecules may be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units,diamines, diacids, amino acids, or combinations thereof. Other linkermolecules may be used in light of this disclosure.

[0109] According to alternative embodiments, the linker molecules areselected based upon their hydrophilic/hydrophobic properties to improvepresentation of synthesized polymers to certain receptors. For example,in the case of a hydrophilic receptor, hydrophilic linker molecules willbe preferred so as to permit the receptor to more closely approach thesynthesized polymer.

[0110] According to another alternative embodiment, linker molecules arealso provided with a photocleavable group at an intermediate position.The photocleavable group is preferably cleavable at a wavelengthdifferent from the protective group. This enables removal of the variouspolymers following completion of the synthesis by way of exposure to thedifferent wavelengths of light.

[0111] The linker molecules can be attached to the substrate viacarbon-carbon bonds using, for example, (poly)trifluorochloroethylenesurfaces, or preferably, by siloxane bonds (using, for example, glass orsilicon oxide surfaces). Siloxane bonds with the surface of thesubstrate may be formed in one embodiment via reactions of linkermolecules bearing trichlorosilyl groups. The linker molecules mayoptionally be attached in an ordered array, i.e., as parts of the headgroups in a polymerized Langmuir Blodgett film. In alternativeembodiments, the linker molecules are adsorbed to the surface of thesubstrate.

[0112] The linker molecules and monomers used herein are provided with afunctional group to which is bound a protective group. Preferably, theprotective group is on the distal or terminal end of the linker moleculeopposite the substrate. The protective group may be either a negativeprotective group (i.e., the protective group renders the linkermolecules less reactive with a monomer upon exposure) or a positiveprotective group (i.e., the protective group renders the linkermolecules more reactive with a monomer upon exposure). In the case ofnegative protective groups an additional step of reactivation will berequired. In some embodiments, this will be done by heating.

[0113] The protective group on the linker molecules may be selected froma wide variety of positive light-reactive groups preferably includingnitro aromatic compounds such as o-nitrobenzyl derivatives orbenzylsulfonyl. In a preferred embodiment, 6-nitroveratryloxycarbonyl(NVOC), 2-nitrobenzyloxycarbonyl (NBOC) orα,α-dimethyl-dimethoxybenzyloxycarbonyl (DDZ) is used. In oneembodiment, a nitro aromatic compound containing a benzylic hydrogenortho to the nitro group is used, i.e., a chemical of the form:

[0114] where R₁ is alkoxy, alkyl, halo, aryl, alkenyl, or hydrogen; R₂is alkoxy, alkyl, halo, aryl, nitro, or hydrogen; R₃ is alkoxy, alkyl,halo, nitro, aryl, or hydrogen; R₄ is alkoxy, alkyl, hydrogen, aryl,halo, or nitro; and R₅ is alkyl, alkynyl, cyano, alkoxy, hydrogen, halo,aryl, or alkenyl. Other materials which may be used includeo-hydroxy-α-methyl cinnamoyl derivatives. Photoremovable protectivegroups are described in, for example, Patchornik, J. Am. Chem. Soc.(1970) 92:6333 and Amit et al., J. Org. Chem. (1974) 39:192, both ofwhich are incorporated herein by reference.

[0115] In an alternative embodiment the positive reactive group isactivated for reaction with reagents in solution. For example, a5-bromo-7-nitro indoline group, when bound to a carbonyl, undergoesreaction upon exposure to light at 420 nm.

[0116] In a second alternative embodiment, the reactive group on thelinker molecule is selected from a wide variety of negativelight-reactive groups including a cinammate group.

[0117] Alternatively, the reactive group is activated or deactivated byelectron beam lithography, x-ray lithography, or any other radiation.Suitable reactive groups for electron beam lithography include sulfonyl.Other methods may be used including, for example, exposure to a currentsource. Other reactive groups and methods of activation may be used inlight of this disclosure.

[0118] As shown in FIG. 1, the linking molecules are preferably exposedto, for example, light through a suitable mask 8 using photolithographictechniques of the type known in the semiconductor industry and describedin, for example, Sze, VLSI Technology, McGraw-Hill (1983), and Mead etal., Introduction to VLSI Systems, Addison-Wesley (1980), which areincorporated herein by reference for all purposes. The light may be,directed at either the surface containing the protective groups or atthe back of the substrate, so long as the substrate is transparent tothe wavelength of light needed for removal of the protective groups. Inthe embodiment shown in FIG. 1, light is directed at the surface of thesubstrate containing the protective groups. FIG. 1 illustrates the useof such masking techniques as they are applied to a positive reactivegroup so as to activate linking molecules and expose functional groupsin areas 10 a and 10 b.

[0119] The mask 8 is in one embodiment a transparent support materialselectively coated with a layer of opaque material. Portions of theopaque material are removed, leaving opaque material in the precisepattern desired on the substrate surface. The mask is brought into closeproximity with, imaged on, or brought directly into contact with thesubstrate surface as shown in FIG. 1. “Openings” in the mask correspondto locations on the substrate where it is desired to removephotoremovable protective groups from the substrate. Alignment may beperformed using conventional alignment techniques in which alignmentmarks (not shown) are used to accurately overlay successive masks withprevious patterning steps, or more sophisticated techniques may be used.For example, interferometric techniques such as the one described inFlanders et al., “A New Interferometric Alignment Technique,” App. Phys.Lett. (1977) 31:426-428, which is incorporated herein by reference, maybe used.

[0120] To enhance contrast of light applied to the substrate, it isdesirable to provide contrast enhancement materials between the mask andthe substrate according to some embodiments. This contrast enhancementlayer may comprise a molecule which is decomposed by light such asquinone diazid or a material which is transiently bleached at thewavelength of interest. Transient bleaching of materials will allowgreater penetration where light is applied, thereby enhancing contrast.Alternatively, contrast enhancement may be provided by way of a claddedfiber optic bundle.

[0121] The light may be from a conventional incandescent source, alaser, a laser diode, or the like. If non-collimated sources of lightare used it may be desirable to provide a thick- or multi-layered maskto prevent spreading of the light onto the substrate. It may, further,be desirable in some embodiments to utilize groups which are sensitiveto different wavelengths to control synthesis. For example, by usinggroups which are sensitive to different wavelengths, it is possible toselect branch positions in the synthesis of a polymer or eliminatecertain masking steps. Several reactive groups along with theircorresponding wavelengths for deprotection are provided in Table 1.TABLE 1 Approximate Group Deprotection Wavelength Nitroveratryloxycarbonyl (NVOC) UV (300-400 nm) Nitrobenzyloxy carbonyl (NBOC) UV(300-350 nm) Dimethyl dimethoxybenzyloxy carbonyl UV (280-300 nm)5-Bromo-7-nitroindolinyl UV (420 nm) o-Hydroxy-α-methyl cinnamoyl UV(300-350 nm) 2-Oxymethylene anthraquinone UV (350 nm)

[0122] While the invention is illustrated primarily herein by way of theuse of a mask to illuminate selected regions the substrate, othertechniques may also be used. For example, the substrate may betranslated under a modulated laser or diode light source. Suchtechniques are discussed in, for example, U.S. Pat. No. 4,719,615(Feyrer et al.), which is incorporated herein by reference. Inalternative embodiments a laser galvanometric scanner is utilized. Inother embodiments, the synthesis may take place on or in contact with aconventional liquid crystal (referred to herein as a “light valve”) orfiber optic light sources. By appropriately modulating liquid crystals,light may be selectively controlled so as to permit light to contactselected regions of the substrate. Alternatively, synthesis may takeplace on the end of a series of optical fibers to which light isselectively applied. Other means of controlling the location of lightexposure will be apparent to those of skill in the art.

[0123] The substrate may be irradiated either in contact or not incontact with a solution (not shown) and is, preferably, irradiated incontact with a solution. The solution contains reagents to prevent theby-products formed by irradiation from interfering with synthesis of thepolymer according to some embodiments. Such by-products might include,for example, carbon dioxide, nitrosocarbonyl compounds, styrenederivatives, indole derivatives, and products of their photochemicalreactions. Alternatively, the solution may contain reagents used tomatch the index of refraction of the substrate. Reagents added to thesolution may further include, for example, acidic or basic buffers,thiols, substituted hydrazines and hydroxylamines, reducing agents(e.g., NADH) or reagents known to react with a given functional group(e.g., aryl nitroso+glyoxylic acid→aryl formhydroxamate+CO₂).

[0124] Either concurrently with or after the irradiation step, thelinker molecules are washed or otherwise contacted with a first monomer,illustrated by “A” in regions 12 a and 12 b in FIG. 2. The first monomerreacts with the activated functional groups of the linkage moleculeswhich have been exposed to light. The first monomer, which is preferablyan amino acid, is also provided with a photoprotective group. Thephotoprotective group on the monomer may be the same as or differentthan the protective group used in the linkage molecules, and may beselected from any of the above-described protective groups. In oneembodiment, the protective groups for the A monomer is selected from thegroup NBOC and NVOC.

[0125] As shown in FIG. 3, the process of irradiating is thereafterrepeated, with a mask repositioned so as to remove linkage protectivegroups and expose functional groups in regions 14 a and 14 b which areillustrated as being regions which were protected in the previousmasking step. As an alternative to repositioning of the first mask, inmany embodiments a second mask will be utilized. In other alternativeembodiments, some steps may provide for illuminating a common region insuccessive steps. As shown in FIG. 3, it may be desirable to provideseparation between irradiated regions. For example, separation of about1-5 μm may be appropriate to account for alignment tolerances.

[0126] As shown in FIG. 4, the substrate is then exposed to a secondprotected monomer “B,” producing B regions 16 a and 16 b. Thereafter,the substrate is again masked so as to remove the protective groups andexpose reactive groups on A region 12 a and B region 16 b. The substrateis again exposed to monomer B, resulting in the formation of thestructure shown in FIG. 6. The dimers B-A and B-B have been produced onthe substrate.

[0127] A subsequent series of masking and contacting steps similar tothose described above with A (not shown) provides the structure shown inFIG. 7. The process provides all possible dimers of B and A, i.e., B-A,A-B, A-A, and B-B.

[0128] The substrate, the area of synthesis, and the area for synthesisof each individual polymer could be of any size or shape. For example,squares, ellipsoids, rectangles, triangles, circles, or portionsthereof, along with irregular geometric shapes, may be utilized.Duplicate synthesis areas may also be applied to a single substrate forpurposes of redundancy.

[0129] In one embodiment the regions 12 and 16 on the substrate willhave a surface area of between about 1 cm² and 10⁻¹⁰ cm². In someembodiments the regions 12 and 16 have areas of less than about 10⁻¹cm², 10⁻² cm², 10⁻³ cm², 10⁻⁴ cm², 10⁻⁵ cm², 10⁻⁶ cm², 10⁻⁷ cm², 10⁻⁸cm², 10⁻¹⁰ cm². In a preferred embodiment, the regions 12 and 16 arebetween about 10×10 μm and 500×500 μm.

[0130] In some embodiments a single substrate supports more than about10 different monomer sequences and perferably more than about 100different monomer sequences, although in some embodiments more thanabout 10³, 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ different sequences are providedon a substrate. Of course, within a region of the substrate in which amonomer sequence is synthesized, it is preferred that the monomersequence be substantially pure. In some embodiments, regions of thesubstrate contain polymer sequences which are at least about 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, or 99% pure.

[0131] According to some embodiments, several sequences areintentionally provided within a single region so as to provide aninitial screening for biological activity, after which materials withinregions exhibiting significant binding are further evaluated.

[0132] IV. Details of One Embodiment of a Reactor System

[0133]FIG. 8A schematically illustrates a preferred embodiment of areactor system 100 for synthesizing polymers on the prepared substratein accordance with one aspect of the invention. The reactor systemincludes a body 102 with a cavity 104 on a surface thereof. In preferredembodiments the cavity 104 is between about 50 and 1000 μm deep with adepth of about 500 μm preferred.

[0134] The bottom of the cavity is preferably provided with an array ofridges 106 which extend both into the plane of the Figure and parallelto the plane of the Figure. The ridges are preferably about 50 to 200 μmdeep and spaced at about 2 to 3 mm. The purpose of the ridges is togenerate turbulent flow for better mixing. The bottom surface of thecavity is preferably light absorbing so as to prevent reflection ofimpinging light.

[0135] A substrate 112 is mounted above the cavity 104. The substrate isprovided along its bottom surface 114 with a photoremovable protectivegroup such as NVOC with or without an intervening linker molecule. Thesubstrate is preferably transparent to a wide spectrum of light, but insome embodiments is transparent only at a wavelength at which theprotective group may be removed (such as UV in the case of NVOC). Thesubstrate in some embodiments is a conventional microscope glass slideor cover slip. The substrate is preferably as thin as possible, whilestill providing adequate physical support. Preferably, the substrate isless than about 1 mm thick, more preferably less than 0.5 mm thick, morepreferably less than 0.1 mm thick, and most preferably less than 0.05 mmthick. In alternative preferred embodiments, the substrate is quartz orsilicon.

[0136] The substrate and the body serve to seal the cavity except for aninlet port 108 and an outlet port 110. The body and the substrate may bemated for sealing in some embodiments with one or more gaskets.According to a preferred embodiment, the body is provided with twoconcentric gaskets and the intervening space is held at vacuum to ensuremating of the substrate to the gaskets.

[0137] Fluid is pumped through the inlet port into the cavity by way ofa pump 116 which may be, for example, a model no. B-120-S made by EldexLaboratories. Selected fluids are circulated into the cavity by thepump, through the cavity, and out the outlet for recirculation ordisposal. The reactor may be subjected to ultrasonic radiation and/orheated to aid in agitation in some embodiments.

[0138] Above the substrate 112, a lens 120 is provided which may be, forexample, a 2″ 100 mm focal length fused silica lens. For the sake of acompact system, a reflective mirror 122 may be provided for directinglight from a light source 124 onto the substrate. Light source 124 maybe, for example, a Xe(Hg) light source manufactured by Oriel and havingmodel no. 66024. A second lens 126 may be provided for the purpose ofprojecting a mask image onto the substrate in combination with lens 112.This form of lithography is referred to herein as projection printing.As will be apparent from this disclosure, proximity printing and thelike may also be used according to some embodiments.

[0139] Light from the light source is permitted to reach only selectedlocations on the substrate as a result of mask 128. Mask 128 may be, forexample, a glass slide having etched chrome thereon. The mask 128 in oneembodiment is provided with a grid of transparent locations and opaquelocations. Such masks may be manufactured by, for example, PhotoSciences, Inc. Light passes freely through the transparent regions ofthe mask, but is reflected from or absorbed by other regions. Therefore,only selected regions of the substrate are exposed to light.

[0140] As discussed above, light valves (LCD's) may be used as analternative to conventional masks to selectively expose regions of thesubstrate. Alternatively, fiber optic faceplates such as those availablefrom Schott Glass, Inc, may be used for the purpose of contrastenhancement of the mask or as the sole means of restricting the regionto which light is applied. Such faceplates would be placed directlyabove or on the substrate in the reactor shown in FIG. 8A. In stillfurther embodiments, flys-eye lenses, tapered fiber optic faceplates, orthe like, may be used for contrast enhancement.

[0141] In order to provide for illumination of regions smaller than awavelength of light, more elaborate techniques may be utilized. Forexample, according to one preferred embodiment, light is directed at thesubstrate by way of molecular microcrystals on the tip of, for example,micropipettes. Such devices are disclosed in Lieberman et al., “A LightSource Smaller Than the optical Wavelength,” Science (1990) 247:59-61,which is incorporated herein by reference for all purposes.

[0142] In operation, the substrate is placed on the cavity and sealedthereto. All operations in the process of preparing the substrate arecarried out in a room lit primarily or entirely by light of a wavelengthoutside of the light range at which the protective group is removed. Forexample, in the case of NVOC, the room should be lit with a conventionaldark room light which provides little or no UV light. All operations arepreferably conducted at about room temperature.

[0143] A first, deprotection fluid (without a monomer) is circulatedthrough the cavity. The solution preferably is of 5 mm sulfuric acid indioxane solution which serves to keep exposed amino groups protonatedand decreases their reactivity with photolysis by-products. Absorptivematerials such as N,N-diethylamino 2,4-dinitrobenzene, for example, maybe included in the deprotection fluid which serves to absorb light andprevent reflection and unwanted photolysis.

[0144] The slide is, thereafter, positioned in a light raypath from themask such that first locations on the substrate are illuminated and,therefore, deprotected. In preferred embodiments the substrate isilluminated for between about 1 and 15 minutes with a preferredillumination time of about 10 minutes at 10-20 mW/cm² with 365 nm light.The slides are neutralized (i.e., brought to a pH of about 7) afterphotolysis with, for example, a solution of di-isopropylethylamine(DIEA) in methylene chloride for about 5 minutes.

[0145] The first monomer is then placed at the first locations on thesubstrate. After irradiation, the slide is removed, treated in bulk, andthen reinstalled in the flow cell. Alternatively, a fluid containing thefirst monomer, preferably also protected by a protective group, iscirculated through the cavity by way of pump 116. If, for example, it isdesired to attach the amino acid Y to the substrate at the firstlocations, the amino acid Y (bearing a protective group on itsα-nitrogen), along with reagents used to render the monomer reactive,and/or a carrier, is circulated from a storage container 118, throughthe pump, through the cavity, and back to the inlet of the pump.

[0146] The monomer carrier solution is, in a preferred embodiment,formed by mixing of a first solution (referred to herein as solution“A”) and a second solution (referred to herein as solution “B”). Table 2provides an illustration of a mixture which may be used for solution A.TABLE 2 Representative Monomer Carrier Solution “A” 100 mg NVOC aminoprotected amino acid 37 mg HOBT (1-Hydroxybenzotriazole) 250 μl DMF(Dimethylformamide) 86 μl DIEA (Diisopropylethylamine)

[0147] The composition of solution B is illustrated in Table 3.Solutions A and B are mixed and allowed to react at room temperature forabout 8 minutes, then diluted with 2 ml of DMF, and 500 μl are appliedto the surface of the slide or the solution is circulated through thereactor system and allowed to react for about 2 hours at roomtemperature. The slide is then washed with DMF, methylene chloride andethanol. TABLE 3 Representative Monomer Carrier Solution “B” 250 μl DMF111 mg BOP (Benzotriazolyl-n-oxy-tris (dimethylamino)phosphoniumhexafluorophosphate)

[0148] As the solution containing the monomer to be attached iscirculated through the cavity, the amino acid or other monomer willreact at its carboxy terminus with amino groups on the regions of thesubstrate which have been deprotected. Of course, while the invention isillustrated by way of circulation of the monomer through the cavity, theinvention could be practiced by way of removing the slide from thereactor and submersing it in an appropriate monomer solution.

[0149] After addition of the first monomer, the solution containing thefirst amino acid is then purged from the system. After circulation of asufficient amount of the DMF/methylene chloride such that removal of theamino acid can be assured (e.g., about 50× times the volume of thecavity and carrier lines), the mask or substrate is repositioned, or anew mask is utilized such that second regions on the substrate will beexposed to light and the light 124 is engaged for a second exposure.This will deprotect second regions on the substrate and the process isrepeated until the desired polymer sequences have been synthesized.

[0150] The entire derivatized substrate is then exposed to a receptor ofinterest, preferably labeled with, for example, a fluorescent marker, bycirculation of a solution or suspension of the receptor through thecavity or by contacting the surface of the slide in bulk. The receptorwill preferentially bind to certain regions of the substrate whichcontain complementary sequences.

[0151] Antibodies are typically suspended in what is commonly referredto as “supercocktail,” which may be, for example, a solution of about 1%BSA (bovine serum albumin), 0.5% Tween in PBS (phosphate bufferedsaline) buffer. The antibodies are diluted into the supercocktail bufferto a final concentration of, for example, about 0.'to 4 μg/ml.

[0152]FIG. 8B illustrates an alternative preferred embodiment of thereactor shown in FIG. 8A. According to this embodiment, the mask 128 isplaced directly in contact with the substrate. Preferably, the etchedportion of the mask is placed face down so as to reduce the effects oflight dispersion. According to this embodiment, the imaging lenses 120and 126 are not necessary because the mask is brought into closeproximity with the substrate.

[0153] For purposes of increasing the signal-to-noise ratio of thetechnique, some embodiments of the invention provide for exposure of thesubstrate to a first labeled or unlabeled receptor followed by exposureof a labeled, second receptor (e.g., an antibody) which binds atmultiple sites on the first receptor. If, for example, the firstreceptor is an antibody derived from a first species of an animal, thesecond receptor is an antibody derived from a second species directed toepitopes associated with the first species. In the case of a mouseantibody, for example, fluorescently labeled goat antibody or antiserumwhich is antimouse may be used to bind at multiple sites on the mouseantibody, providing several times the fluorescence compared to theattachment of a single mouse antibody at each binding site. This processmay be repeated again with additional antibodies (e.g., goat-mouse-goat,etc.) for further signal amplification.

[0154] In preferred embodiments an ordered sequence of masks isutilized. In some embodiments it is possible to use as few as a singlemask to synthesize all of the possible polymers of a given monomer set.

[0155] If, for example, it is desired to synthesize all 16 dinucleotidesfrom four bases, a 1 cm square synthesis region is divided conceptuallyinto 16 boxes, each 0.25 cm wide. Denote the four monomer units by A, B,C, and D. The first reactions are carried out in four vertical columns,each 0.25 cm wide. The first mask exposes the left-most column of boxes,where A is coupled. The second mask exposes the next column, where B iscoupled; followed by a third mask, for the C column; and a final maskthat exposes the right-most column, for D. The first, second, third, andfourth masks may be a single mask translated to different locations.

[0156] The process is repeated in the horizontal direction for thesecond unit of the dimer. This time, the masks allow exposure ofhorizontal rows, again 0.25 cm wide. A, B, C, and D are sequentiallycoupled using masks that expose horizontal fourths of the reaction area.The resulting substrate contains all 16 dinucleotides of four bases.

[0157] The eight masks used to synthesize the dinucleotide are relatedto one another by translation or rotation. In fact, one mask can be usedin all eight steps if it is suitably rotated and translated. Forexample, in the example above, a mask with a single transparent regioncould be sequentially used to expose each of the vertical columns,translated 90°, and then sequentially used to allow exposure of thehorizontal rows.

[0158] Tables 4 and 5 provide a simple computer program in Quick Basicfor planning a masking program and a sample output, respectively, forthe synthesis of a polymer chain of three monomers (“residues”) havingthree different monomers in the first level, four different monomers inthe second level, and five different monomers in the third level in astriped pattern. The output of the program is the number of cells, thenumber of “stripes” (light regions) on each mask, and the amount oftranslation required for each exposure of the mask. TABLE 4 MaskStrategy Program DEFINT A-Z DIM b(20), w(20), 1(500) F$ = “LPT1:” OPENf$ FOR OUTPUT AS #1 jmax = 3  ′Number of residues b(1) = 3: b(2) = 4:b(3) = 5  ′Number of building blocks for res 1,2,3 g = l: lmax(1) = lFOR j = l TO jmax: g = g * b(j): NEXT j w(0) = 0: w(l) = g / b(l) PRINT#1, “MASK2.BAS ”, DATE$, TIME$: PRINT #1, PRINT #1, USING “Number ofresidues=##“; jmax FOR j = 1 TO jmax PRINT #1, USING “  Residue##  building blocks”; j; b(j) NEXT j PRINT #1, “ PRINT #1, USING “Numberof cells=####”; g: PRINT #1, FOR j = 2 TO jmax lmax(j) = lmax(j − 1) *b(j − l) w(j) = w(j − l) / b(j) NEXT j FOR j = l TO jmax PRINT #1, USING“Mask for residue ##”; j: PRINT #1, PRINT #1, USING “ Number of stripes=###”; lmax(j) PRINT #1, USING “ Width of each stripe =###”; w(j) FOR l= l TO lmax(j) a = 1 +(l − l) * w(j − l) ae = a + w(j) − l PRINT #1,USING “ Stripe ## begins at location ### and ends at ###”; l; a; ae NEXTl PRINT #1, PRINT #1, USING “ For each of ## building blocks, translatemask by ## cell(s)“; b(j); w(j), PRINT #1, : PRINT #1, : PRINT #1, NEXTj

[0159] TABLE 5 Masking Strategy Output Number of residues= 3 Residue 1 3building blocks Residue 2 4 building blocks Residue 3 5 building blocksNumber of cells= 60 Mask for residue 1 Number of stripes = 1 Width ofeach stripe = 20 Stripe 1 begins at location  1 and ends at 20 For eachof 3 building blocks, translate mask by 20 cell(s) Mask for residue 2Number of stripes = 3 Width of each stripe = 5 Stripe 1 begins atlocation 1 and ends at 5 Stripe 2 begins at location 21 and ends at 25Stripe 3 begins at location 41 and ends at 45 For each of 4 buildingblocks, translate mask by 5 cell(s) Mask for residue 3 Number of stripes= 12 Width of each stripe = 1 Stripe 1 begins at location 1 and ends at1 Stripe 2 begins at location 6 and ends at 6 Stripe 3 begins atlocation 11 and ends at 11 Stripe 4 begins at location 16 and ends at 16Stripe 5 begins at location 21 and ends at 21 Stripe 6 begins atlocation 26 and ends at 26 Stripe 7 begins at location 31 and ends at 31Stripe 8 begins at location 36 and ends at 36 Stripe 9 begins atlocation 41 and ends at 41 Stripe 10 begins at location 46 and ends at46 Stripe 11 begins at location 51 and ends at 51 Stripe 12 begins atlocation 56 and ends at 56 For each of 5 building blocks, translate maskby 1 cell(s)

[0160] V. Details of One Embodiment of A Fluorescent Detection Device

[0161]FIG. 9 illustrates a fluorescent detection device for detectingfluorescently labeled receptors on a substrate. A substrate 112 isplaced on an x/y translation table 202. In a preferred embodiment thex/y translation table is a model no. PM500-A1 manufactured by NewportCorporation. The x/y translation table is connected to and controlled byan appropriately programmed digital computer 204 which may be, forexample, an appropriately programmed IBM PC/AT or AT compatiblecomputer. Of course, other computer systems, special purpose hardware,or the like could readily be substituted for the AT computer used hereinfor illustration. Computer software for the translation and datacollection functions described herein can be provided based oncommercially available software including, for example, “Lab Windows”licensed by National Instruments, which is incorporated herein byreference for all purposes.

[0162] The substrate and x/y translation table are placed under amicroscope 206 which includes one or more objectives 208. Light (about488 nm) from a laser 210, which in some embodiments is a model no.2020-05 argon ion laser manufactured by Spectraphysics, is directed atthe substrate by a dichzoic mirror 207 which passes greater than about520 nm light but reflects 488 nm light. Dichroic mirror 207 may be, forexample, a model no. FT510 manufactured by Carl Zeiss. Light reflectedfrom the mirror then enters the microscope 206 which may be, forexample, a model no. Axioscop 20 manufactured by Carl Zeiss.Fluorescein-marked materials on the substrate will fluoresce >488 nmlight, and the fluoresced light will be collected by the microscope andpassed through the mirror. The fluorescent light from the substrate isthen directed through a wavelength filter 209 and, thereafter through anaperture plate 211. Wavelength filter 209 may be, for example, a modelno. OG530 manufactured by Melles Griot and aperture plate 211 may be,for example, a model no. 477352/477380 manufactured by Carl Zeiss.

[0163] The fluoresced light then enters a photomultiplier tube 212 whichin some embodiments is a model no. R943-02 manufactured by Hamamatsu,the signal is amplified in preamplifier 214 and photons are counted byphoton counter 216. The number of photons is recorded as a function ofthe location in the computer 204. Pre-Amp 214 may be, for example, amodel no. SR440 manufactured by Stanford Research Systems and photoncounter 216 may be a model no. SR400 manufactured by Stanford ResearchSystems. The substrate is then moved to a subsequent location and theprocess is repeated. In preferred embodiments the data are acquiredevery 1 to 100 μm with a data collection diameter of about 0.8 to 10 μmpreferred. In embodiments with sufficiently high fluorescence, a CCDdetector with broadfield illumination is utilized.

[0164] By counting the number of photons generated in a given area inresponse to the laser, it is possible to determine where fluorescentmarked molecules are located on the substrate. Consequently, for a slidewhich has a matrix of polypeptides, for example, synthesized on thesurface thereof, it is possible to determine which of the polypeptidesis complementary to a fluorescently marked receptor.

[0165] According to preferred embodiments, the intensity and duration ofthe light applied to the substrate is controlled by varying the laserpower and scan stage rate for improved signal-to-noise ratio bymaximizing fluorescence emission and minimizing background noise.

[0166] While the detection apparatus has been illustrated primarilyherein with regard to the detection of marked receptors, the inventionwill find application in other areas. For example, the detectionapparatus disclosed herein could be used in the fields of catalysis, DNAor protein gel scanning, and the like.

[0167] VI. Determination of Relative Binding Strength of Receptors

[0168] The signal-to-noise ratio of the present invention issufficiently high that not only can the presence or absence of areceptor on a ligand be detected, but also the relative binding affinityof receptors to a variety of sequences can be determined.

[0169] In practice it is found that a receptor will bind to severalpeptide sequences in an array, but will bind much more strongly to somesequences than others. Strong binding affinity will be evidenced hereinby a strong fluorescent or radiographic signal since many receptormolecules will bind in a region of a strongly bound ligand. Conversely,a weak binding affinity will be evidenced by a weak fluorescent orradiographic signal due to the relatively small number of receptormolecules which bind in a particular region of a substrate having aligand with a weak binding affinity for the receptor. Consequently, itbecomes possible to determine relative binding avidity (or affinity inthe case of univalent interactions) of a ligand herein by way of theintensity of a fluorescent or radiographic signal in a region containingthat ligand.

[0170] Semiquantitative data on affinities might also be obtained byvarying washing conditions and concentrations of the receptor. Thiswould be done by comparison to known ligand receptor pairs, for example.

VII. EXAMPLES

[0171] The following examples are provided to illustrate the efficacy ofthe inventions herein. All operations were conducted at about ambienttemperatures and pressures unless indicated to the contrary.

[0172] A. Slide Preparation

[0173] Before attachment of reactive groups it is preferred to clean thesubstrate which is, in a preferred embodiment a glass substrate such asa microscope slide or cover slip. According to one embodiment the slideis soaked in an alkaline bath consisting of, for example, 1 liter of 95%ethanol with 120 ml of water and 120 grams of sodium hydroxide for 12hours. The slides are then washed under running water and allowed to airdry, and rinsed once with a solution of 95% ethanol.

[0174] The slides are then aminated with, for example,aminopropyltriethoxysilane for the purpose of attaching amino groups tothe glass surface on linker molecules, although any omega functionalizedsilane could also be used for this purpose. In one embodiment 0.1%aminopropyltriethoxysilane is utilized, although solutions withconcentrations from 10⁻⁷% to 10% may be used, with about 10⁻³% to 2%preferred. A 0.1% mixture is prepared by adding to 100 ml of a 95%ethanol/5% water mixture, 100 microliters (μl) ofaminopropyltriethoxysilane. The mixture is agitated at about ambienttemperature on a rotary shaker for about 5 minutes. 500 μl of thismixture is then applied to the surface of one side of each cleanedslide. After 4 minutes, the slides are decanted of this solution andrinsed three times by dipping in, for example, 100% ethanol.

[0175] After the plates dry, they are placed in a 110-120° C. vacuumoven for about 20 minutes, and then allowed to cure at room temperaturefor about 12 hours in an argon environment. The slides are then dippedinto DMF (dimethylformamide) solution, followed by a thorough washingwith methylene chloride.

[0176] The aminated surface of the slide is then exposed to about 500 μlof, for example, a 30 millimolar (mM) solution of NVOC-GABA (gamma aminobutyric acid) NHS (N-hydroxysuccinimide) in DMF for attachment of aNVOC-GABA to each of the amino groups.

[0177] The surface is washed with, for example, DMF, methylene chloride,and ethanol.

[0178] Any unreacted aminopropyl silane on the surface—that is, thoseamino groups which have not had the NVOC-GABA attached—are now cappedwith acetyl groups (to prevent further reaction) by exposure to a 1:3mixture of acetic anhydride in pyridine for 1 hour. Other materialswhich may perform this residual capping function include trifluoroaceticanhydride, formicacetic anhydride, or other reactive acylating agents.Finally, the slides are washed again with DMF, methylene chloride, andethanol.

[0179] B. Synthesis of Eight Trimers of “A” and “B”

[0180]FIG. 10 illustrates a possible synthesis of the eight trimers ofthe two-monomer set: gly, phe (represented by “A” and “B,”respectively). A glass slide bearing silane groups terminating in6-nitroveratryloxycarboxamide (NVOC—NH) residues is prepared as asubstrate. Active esters (pentafluorophenyl, OBt, etc.) of gly and pheprotected at the amino group with NVOC are prepared as reagents. Whilenot pertinent to this example, if side chain protecting groups arerequired for the monomer set, these must not be photoreactive at thewavelength of light used to protect the primary chain.

[0181] For a monomer set of size n, n×l cycles are required tosynthesize all possible sequences of length l. A cycle consists of:

[0182] 1. Irradiation through an appropriate mask to expose the aminogroups at the sites where the next residue is to be added, withappropriate washes to remove the by-products of the deprotection.

[0183] 2. Addition of a single activated and protected (with the samephotochemically-removable group) monomer, which will react only at thesites addressed in step 1, with appropriate washes to remove the excessreagent from the surface.

[0184] The above cycle is repeated for each member of the monomer setuntil each location on the surface has been extended by one residue inone embodiment. In other embodiments, several residues are sequentiallyadded at one location before moving on to the next location. Cycle timeswill generally be limited by the coupling reaction rate, now as short as20 min in automated peptide synthesizers. This step is optionallyfollowed by addition of a protecting group to stabilize the array forlater testing. For some types of polymers (e.g., peptides), a finaldeprotection of the entire surface (removal of photoprotective sidechain groups) may be required.

[0185] More particularly, as shown in FIG. 10A, the glass 20 is providedwith regions 22, 24, 26, 28, 30, 32, 34, and 36. Regions 30, 32, 34, and36 are masked, as shown in FIG. 10B and the glass is irradiated andexposed to a reagent containing “A” (e.g., gly), with the resultingstructure shown in FIG. 10C. Thereafter, regions 22, 24, 26, and 28 aremasked, the glass is irradiated (as shown in FIG. 10D) and exposed to areagent containing “B” (e.g., phe), with the resulting structure shownin FIG. 10E. The process proceeds, consecutively masking and exposingthe sections as shown until the structure shown in FIG. 10M is obtained.The glass is irradiated and the terminal groups are, optionally, cappedby acetylation. As shown, all possible trimers of gly/phe are obtained.

[0186] In this example, no side chain protective group removal isnecessary. If it is desired, side chain deprotection may be accomplishedby treatment with ethanedithiol and trifluoroacetic acid.

[0187] In general, the number of steps needed to obtain a particularpolymer chain is defined by:

n×l  (1)

[0188] where:

[0189] n=the number of monomers in the basis set of monomers, and

[0190] l=the number of monomer units in a polymer chain.

[0191] Conversely, the synthesized number of sequences of length l willbe:

n^(l).  (2)

[0192] Of course, greater diversity is obtained by using maskingstrategies which will also include the synthesis of polymers having alength of less than l. If, in the extreme case, all polymers having alength less than or equal to t are synthesized, the number of polymerssynthesized will be:

n^(l)+n^(l−1)+ . . . +n¹.  (3)

[0193] The maximum number of lithographic steps needed will generally ben for each “layer” of monomers, i.e., the total number of masks (and,therefore, the number of lithographic steps) needed will be n×l. Thesize of the transparent mask regions will vary in accordance with thearea of the substrate available for synthesis and the number ofsequences to be formed. In general, the size of the synthesis areas willbe:

size of synthesis areas=(A)/(S)

[0194] where:

[0195] A is the total area available for synthesis; and

[0196] S is the number of sequences desired in the area.

[0197] It will be appreciated by those of skill in the art that theabove method could readily be used to simultaneously produce thousandsor millions of oligomers on a substrate using the photolithographictechniques disclosed herein. Consequently, the method results in theability to practically test large numbers of, for example, di, tri,tetra, penta, hexa, hepta, octapeptides, dodecapeptides, or largerpolypeptides (or correspondingly, polynucleotides).

[0198] The above example has illustrated the method by way of a manualexample. It will of course be appreciated that automated orsemi-automated methods could be used. The substrate would be mounted ina flow cell for automated addition and removal of reagents, to minimizethe volume of reagents needed, and to more carefully control reactionconditions. Successive masks could be applied manually or automatically.

[0199] C. Synthesis of a Dimer of an Aminopropyl Group and a FluorescentGroup

[0200] In synthesizing the diner of an aminopropyl group and afluorescent group, a functionalized durapore membrane was used assubstrate. The durapore membrane was a polyvinylidine difluoride withaminopropyl groups. The aminopropyl groups were protected with the DDZgroup by reaction of the carbonyl chloride with the amino groups, areaction readily known to those of skill in the art. The surface bearingthese groups was placed in a solution of THF and contacted with a maskbearing a checkerboard pattern of 1 mm opaque and transparent regions.The mask was exposed to ultraviolet light having a wavelength down to atleast about 280 nm for about 5 minutes at ambient temperature, althougha wide range of exposure times and temperatures may be appropriate invarious embodiments of the invention. For example, in one embodiment, anexposure time of between about 1 and 5000 seconds may be used at processtemperatures of between −70 and +50°C.

[0201] In one preferred embodiment, exposure times of between about 1and 500 seconds at about ambient pressure are used. In some preferredembodiments, pressure above ambient is used to prevent evaporation.

[0202] The surface of the membrane was then washed for about 1 hour witha fluorescent label which included an active ester bound to a chelate ofa lanthanide. Wash times will vary over a wide range of values fromabout a few minutes to a few hours. These materials fluoresce in the redand the green visible region. After the reaction with the active esterin the fluorophore was complete, the locations in which the fluorophorewas bound could be visualized by exposing them to ultraviolet light andobserving the red and the green fluorescence. It was observed that thederivatized regions of the substrate closely corresponded to theoriginal pattern of the mask.

[0203] D. Demonstration of Signal Capability

[0204] Signal detection capability was demonstrated using a low-levelstandard fluorescent bead kit manufactured by Flow Cytometry Standardaand having model no. 824. This kit includes 5.8 μm diameter beads, eachimpregnated with a known number of fluorescein molecules.

[0205] One of the beads was placed in the illumination field on the scanstage as shown in FIG. 9 in a field of a laser spot which was initiallyshuttered. After being positioned in the illumination field, the photondetection equipment was turned on. The laser beam was unblocked and itinteracted with the particle bead, which then fluoresced. Fluorescencecurves of beads impregnated with 7,000; 13,000; and 29,000 fluoresceinmolecules, are shown in FIGS. 11A, 11B, and 11C respectively. On eachcurve, traces for beads without fluorescein molecules are also shown.These experiments were performed with 488 nm excitation, with 100 μW oflaser power. The light was focused through a 40 power 0.75 NA objective.

[0206] The fluorescence intensity in all cases started off at a highvalue and then decreased exponentially. The fall-off in intensity is dueto photobleaching of the fluorescein molecules. The traces of beadswithout fluorescein molecules are used for background subtraction. Thedifference in the initial exponential decay between labeled andnonlabeled beads is integrated to give the total number of photoncounts, and this number is related to the number of molecules per bead.Therefore, it is possible to deduce the number of photons perfluorescein molecule that can be detected. For the curves illustrated inFIG. 11, this calculation indicates the radiation of about 40 to 50photons per fluorescein molecule are detected.

[0207] E. Determination of the Number of Molecules Per Unit Area

[0208] Aminopropylated glass microscope slides prepared according to themethods discussed above were utilized in order to establish the densityof labeling of the slides. The free amino termini of the slides werereacted with FITC (fluorescein isothiocyanate) which forms a covalentlinkage with the amino group. The slide is then scanned to count thenumber of fluorescent photons generated in a region which, using theestimated 40-50 photons per fluorescent molecule, enables thecalculation of the number of molecules which are on the surface per unitarea.

[0209] A slide with aminopropyl silane on its surface was immersed in a1 mM solution of FITC in DMF for 1 hour at about ambient temperature.After reaction, the slide was washed twice with DMF and then washed withethanol, water, and then ethanol again. It was then dried and stored inthe dark until it was ready to be examined.

[0210] Through the use of curves similar to those shown in FIG. 11, andby integrating the fluorescent counts under the exponentially decayingsignal, the number of free amino groups on the surface afterderivitization was determined. It was determined that slides withlabeling densities of 1 fluoroscein per 10³×10³ to ˜2×2 nm could bereproducibly made as the concentration of aminopropyltriethoxysilanevaried from 10⁻⁵% to 10⁻¹%.

[0211] F. Removal of NVOC and Attachment of A Fluorescent Marker

[0212] NVOC-GABA groups were attached as described above. The entiresurface of one slide was exposed to light so as to expose a free aminogroup at the end of the gamma amino butyric acid. This slide, and aduplicate which was not exposed, were then exposed to fluoresceinisothiocyanate (FITC).

[0213]FIG. 12A illustrates the slide which was not exposed to light, butwhich was exposed to FITC. The units of the x axis are time and theunits of the y axis are counts. The trace contains a certain amount ofbackground fluorescence. The duplicate slide was exposed to 350 nmbroadband illumination for about 1 minute (12 mW/cm², ˜350 nmillumination), washed and reacted with FITC. The fluorescence curves forthis slide are shown in FIG. 12B. A large increase in the level offluorescence is observed, which indicates photolysis has exposed anumber of amino groups on the surface of the slides for attachment of afluorescent marker.

[0214] G. Use of a Mask in Removal of NVOC

[0215] The next experiment was performed with a 0.1% aminopropylatedslide. Light from a Hg—Xe arc lamp was imaged onto the substrate througha laser-ablated chrome-on-glass mask in direct contact with thesubstrate.

[0216] This slide was illuminated for approximately 5 minutes, with 12mW of 350 nm broadband light and then reacted with the 1 mM FITCsolution. It was put on the laser detection scanning stage and a graphwas plotted as a two-dimensional representation of position color-codedfor fluorescence intensity. The fluorescence intensity (in counts) as afunction of location is given on the color scale to the right of FIG.13A for a mask having 100×100 μm squares.

[0217] The experiment was repeated a number of times through variousmasks. The fluorescence pattern for a 50 μm mask is illustrated in FIG.13B, for a 20 μm mask in FIG. 13C, and for a 10 μm mask in FIG. 13D. Themask pattern is distinct down to at least about 10 μm squares using thislithographic technique.

[0218] H. Attachment of YGGFL and Subsequent Exposure to Herz Antibodyand Goat Antimouse

[0219] In order to establish that receptors to a particular polypeptidesequence would bind to a surface-bound peptide and be detected, Leuenkephalin was coupled to the surface and recognized by an antibody. Aslide was derivatized with 0.1% amino propyl-triethoxysilane andprotected with NVOC. A 500 μm checkerboard mask was used to expose theslide in a flow cell using backside contact printing. The Leu enkephalinsequence (H₂N-tyrosine, glycine, glycine; phenylalanine, leucine-CO₂H,otherwise referred to herein as YGGFL) was attached via its carboxy endto the exposed amino groups on the surface of the slide. The peptide wasadded in DMF solution with the BOP/HOBT/DIEA coupling reagents andrecirculated through the flow cell for 2 hours at room temperature.

[0220] A first antibody, known as the Herz antibody, was applied to thesurface of the slide for 45 minutes at 2 μg/ml in a supercocktail(containing 1% BSA and 1% ovalbumin also in this case). A secondantibody, goat anti-mouse fluorescein conjugate, was then added at 2μg/ml in the supercocktail buffer, and allowed to incubate for 2 hours.

[0221] The results of this experiment are provided in FIG. 14. Again,this figure illustrates fluorescence intensity as a function ofposition. The fluorescence scale is shown on the right, according to thecolor coding. This image was taken at 10 μm steps. This figure indicatesthat not only can deprotection be carried out in a well defined pattern,but also that (1) the method provides for successful coupling ofpeptides to the surface of the substrate, (2) the surface of a boundpeptide is available for binding with an antibody, and (3) that thedetection apparatus capabilities are sufficient to detect binding of areceptor.

[0222] I. Monomer-by-Monomer Formation of YGGFL and Subsequent Exposureto Labeled Antibody

[0223] Monomer-by-monomer synthesis of YGGFL and GGFL in alternatesquares was performed on a slide in a checkerboard pattern and theresulting slide was exposed to the Herz antibody. This experiment andthe results thereof are illustrated in FIGS. 15A, 15B, 15C, and 15D.

[0224] In FIG. 15A, a slide is shown which is derivatized with theaminopropyl group, protected in this case with t-BOC (t-butoxycarbonyl).The slide was treated with TFA to remove the t-BOC protecting group.E-aminocaproic acid, which was t-BOC protected at its amino group, wasthen coupled onto the aminopropyl groups. The aminocaproic acid servesas a spacer between the aminopropyl group and the peptide to besynthesized. The amino end of the spacer was deprotected and coupled toNVOC-leucine. The entire slide was then illuminated with 12 mW of 325 nmbroadband illumination. The slide was then coupled withNVOC-phenylalanine and washed. The entire slide was again illuminated,then coupled to NVOC-glycine and washed. The slide was again illuminatedand coupled to NVOC-glycine to form the sequence shown in the lastportion of FIG. 15A.

[0225] As shown in FIG. 15B, alternating regions of the slide were thenilluminated using a projection print using a 500×500 μm checkerboardmask; thus, the amino group of glycine was exposed only in the lightedareas. When the next coupling chemistry step was carried out,NVOC-tyrosine was added, and it coupled only at those spots which hadreceived illumination. The entire slide was then illuminated to removeall the NVOC groups, leaving a checkerboard of YGGFL in the lightedareas and in the other areas, GGFL. The Herz antibody (which recognizesthe YGGFL, but not GGFL) was then added, followed by goat anti-mousefluorescein conjugate.

[0226] The resulting fluorescence scan is shown in FIG. 15C, and thecolor coding for the fluorescence intensity is again given on the right.Dark areas contain the tetrapeptide GGFL, which is not recognized by theHerz antibody (and thus there is no binding of the goat anti-mouseantibody with fluorescein conjugate, and in the red areas YGGFL ispresent. The YGGFL pentapeptide is recognized by the Herz antibody and,therefore, there is antibody in the lighted regions for thefluorescein-conjugated goat anti-mouse to recognize.

[0227] Similar patterns are shown for a 50 μm mask used in directcontact (“proximity print”) with the substrate in FIG. 15D. Note thatthe pattern is more distinct and the corners of the checkerboard patternare touching when the mask is placed in direct contact with thesubstrate (which reflects the increase in resolution using thistechnique).

[0228] J. Monomer-by-Monomer Synthesis of YGGFL and PGGFL

[0229] A synthesis using a 50 μm checkerboard mask similar to that shownin FIG. 15 was conducted. However, P was added to the GGFL sites on thesubstrate through an additional coupling step. P was added by exposingprotected GGFL to light through a mask, and subsequence exposure to P inthe manner set forth above. Therefore, half of the regions on thesubstrate contained YGGFL and the remaining half containe PGGFL.

[0230] The fluorescence plot for this experiment is provided in FIG. 16.As shown, the regions are again readily discernable. This experimentdemonstrates that antibodies are able to recognize a specific sequenceand that the recognition is not length-dependent.

[0231] K. Monomer-by-Monomer Synthesis of YGGFL and YPGGFL

[0232] In order to further demonstrate the operability of the invention,a 50 μm checkerboard pattern of alternating YGGFL and YPGGFL wassynthesized on a substrate using techniques like those set forth above.The resulting fluorescence plot is provided in FIG. 17. Again, it isseen that the antibody is clearly able to recognize the YGGFL sequenceand does not bind significantly at the YPGGF regions.

[0233] L. Synthesis of an Array of Sixteen Different Amino AcidSequences and Estimation of Relative Binding Affinity to Herz Antibody

[0234] Using techniques similar to those set forth above, an array of 16different amino acid sequences (replicated four times) was synthesizedon each of two glass substrates. The sequences were synthesized byattaching the sequence NVOC-GFL across the entire surface of the slides.Using a series of masks, two layers of amino acids were then selectivelyapplied to the substrate. Each region had dimensions of 0.25 cm×0.0625cm. The first slide contained amino acid sequences containing only Lamino acids while the second slide contained selected D amino acids.FIGS. 18A and 18B illustrate a map of the various regions on the firstand second slides, respectively. The patterns shown in FIGS. 18A and 18Bwere duplicated four times on each slide. The slides were then-exposedto the Herz antibody and fluorescein-labeled goat anti-mouse.

[0235]FIG. 19 is a fluorescence plot of the first slide, which containedonly L amino acids. Red indicates strong binding (149,000 counts ormore) while black indicates little or no binding of the Herz antibody(20,000 counts or less). The bottom right-hand portion of the slideappears “cut off” because the slide was broken during processing. Thesequence YGGFL is clearly most strongly recognized. The sequences YAGFLand YSGFL also exhibit strong recognition of the antibody. By contrast,most of the remaining sequences show little or no binding. The fourduplicate portions of the slide are extremely consistent in the amountof binding shown therein.

[0236]FIG. 20 is a fluorescence plot of the second slide. Again,strongest binding is exhibited by the YGGFL sequence. Significantbinding is also detected to YaGFL, YsGFL, and YpGFL. The remainingsequences show less binding with the antibody. Note the low bindingefficiency of the sequence yGGFL.

[0237] Table 6 lists the various sequences tested in order of relativefluorescence, which provides information regarding relative bindingaffinity. TABLE 6 Apparent Binding to Herz Ab L-a.a. Set D-a.a. SetYGGFL YGGFL YAGFL YaGFL YSGFL YsGFL LGGFL YpGFL FGGFL fGGFL YPGFL yGGFLLAGFL faGFL FAGFL wGGFL WGGFL yaGFL fpGFL waGFL

[0238] VIII. Illustrative Alternative Embodiment

[0239] According to an alternative embodiment of the invention, themethods provide for attaching to the surface a caged binding memberwhich in its caged form has a relatively low affinity for otherpotentially binding species, such as receptors and specific bindingsubstances. Such techniques are more fully described in co-pendingapplication Ser. No. 404,920, filed Sep. 8, 1989, and incorporatedherein by reference for all purposes.

[0240] According to this alternative embodiment, the invention providesmethods for forming predefined regions on a surface of a solid support,wherein the predefined regions are capable of immobilizing receptors.The methods make use of caged binding members attached to the surface toenable selective activation of the predefined regions. The caged bindingmembers are liberated to act as binding members ultimately capable ofbinding receptors upon selective activation of the predefined regions.The activated binding members are then used to immobilize specificmolecules such as receptors on the predefined region of the surface. Theabove procedure is repeated at the same or different sites on thesurface so as to provide a surface prepared with a plurality of regionson the surface containing, for example, the same or different receptors.When receptors immobilized in this way have a differential affinity forone or more ligands, screenings and assays for the ligands can beconducted in the regions of the surface containing the receptors.

[0241] The alternative embodiment may make use of novel caged bindingmembers attached to the substrate. Caged (unactivated) members have arelatively low is affinity for receptors of substances that specificallybind to uncaged binding members when compared with the correspondingaffinities of activated binding members. Thus, the binding members areprotected from reaction until a suitable source of energy is applied tothe regions of the surface desired to be activated. Upon application ofa suitable energy source, the caging groups labilize, thereby presentingthe activated binding member. A typical energy source will be light.

[0242] Once the binding members on the surface are activated they may beattached to a receptor. The receptor chosen may be a monoclonalantibody, a nucleic acid sequence, a drug receptor, etc. The receptorwill usually, though not always, be prepared so as to permit attachingit, directly or indirectly, to a binding member. For example, a specificbinding substance having a strong binding affinity for the bindingmember and a strong affinity for the receptor or a conjugate of thereceptor may be used to act as a bridge between binding members andreceptors if desired. The method uses a receptor prepared such that thereceptor retains its activity toward a particular ligand.

[0243] Preferably, the caged binding member attached to the solidsubstrate will be a photoactivatable biotin complex, i.e., a biotinmolecule that has been chemically modified with photoactivatableprotecting groups so that it has a significantly reduced bindingaffinity for avidin or avidin analogs than does natural biotin. In apreferred embodiment, the protecting groups localized in a predefinedregion of the surface will be removed upon application of a suitablesource of radiation to give binding members, that are biotin or afunctionally analogous compound having substantially the same bindingaffinity for avidin or avidin analogs as does biotin.

[0244] In another preferred embodiment, avidin or an avidin analog isincubated with activated binding members on the surface until the avidinbinds strongly to the binding members. The avidin so immobilized onpredefined regions of the surface can then be incubated with a desiredreceptor or conjugate of a desired receptor. The receptor willpreferably be biotinylated, e.g., a biotinylated antibody, when avidinis immobilized on the predefined regions of the surface. Alternatively,a preferred embodiment will present an avidin/biotinylated receptorcomplex, which has been previously prepared, to activated bindingmembers on the surface.

IX. CONCLUSION

[0245] The present inventions provide greatly improved methods andapparatus for synthesis of polymers on substrates. It is to beunderstood that the above description is intended to be illustrative andnot restrictive. Many embodiments will be apparent to those of skill inthe art upon reviewing the above description. By way of example, theinvention has been described primarily with reference to the use ofphotoremovable protective groups, but it will be readily recognized bythose of skill in the art that sources of radiation other than lightcould also be used. For example, in some embodiments it may be desirableto use protective groups which are sensitive to electron beamirradiation, x-ray irradiation, in combination with electron beamlithograph, or x-ray lithography techniques. Alternatively, the groupcould be removed by exposure to an electric current. The scope of theinvention should, therefore, be determined not with reference to theabove description, but should instead be determined with reference tothe appended claims, along with the full scope of equivalents to whichsuch claims are entitled.

What is claimed is:
 1. A method of preparing sequences on a substratecomprising the steps of: a) exposing a first region of said substrate toan activator to remove a protective group; b) exposing at least saidfirst region to a first monomer; c) exposing a second region to anactivator to remove a protective group; and d) exposing at least saidsecond region to a second monomer.
 2. The method as recited in claim 1wherein said steps of exposing to an activator use an activator selectedfrom the group consisting of ion beams, electron beams, gamma rays,x-rays, ultra-violet radiation, light, infra-red radiation, microwaves,electric currents, radiowaves, and combinations thereof.
 3. The methodas recited in claim 1 wherein said protective groups are photosensitiveprotective groups.
 4. The method as recited in claim 1 wherein saidsteps of exposing to an activator are steps of applying light toselected regions of said substrate.
 5. The method as recited in claim 1wherein said first and the second monomers are amino acids.
 6. Themethod as recited in claim 1 further comprising a step of screeningsequences on said substrate for affinity with a receptor, said step ofscreening further comprising the step of exposing said substrate to saidreceptor and testing for the presence of said receptor in said first andsaid second region.
 7. The method as recited in claim 6 wherein saidreceptor is an antibody.
 8. The method as recited in claim 1 whereinsaid substrate is selected from the group consisting of polymerizedLangmuir Blodgett film, functionalized glass, germanium, silicon,polymers, (poly)tetrafluoroethylene, polystyrene, gallium arsenide, andcombinations thereof.
 9. The method as recited in claim 1 wherein saidprotective group is selected from the group consisting ofortho-nitrobenzyl derivatives, 6-nitroveratryloxycarbonyl,2-nitrobenzyloxycarbonyl, cinnamoyl derivatives, and mixtures thereof.10. The method as recited in claim 1 wherein said first and secondregions each have total areas of less than 1 cm².
 11. The method asrecited in claim 1 wherein said first and second regions each have totalareas of between about 1 μm² and 10,000 μm².
 12. The method as recitedin claim 4 wherein said light is monochromatic coherent light.
 13. Themethod as recited in claim 1 wherein said steps of exposing to anactivator are carried out with a solution in contact with saidsubstrate.
 14. The method as recited in claim 13 wherein said solutionfurther comprises said first or said second monomer.
 15. The method asrecited in claim 6 wherein said receptor further comprises a markerselected from the group consisting of radioactive markers andfluorescent markers and wherein said step of testing for the presence ofthe receptor is a step of detecting said marker.
 16. The method asrecited in claim 1 wherein the steps of exposing to an activator furthercomprise steps of: a) placing a mask adjacent to said substrate, saidmask having substantially transparent regions and substantially opaqueregions at a wavelength of light; and b) illuminating said mask with alight source, said light source producing at least said wavelength oflight.
 17. The method as recited in claim 1 wherein said steps arerepeated so as to synthesize 10³ or more different sequences on saidsubstrate.
 18. The method as recited in claim 1 wherein said steps arerepeated so as to synthesize 10⁶ or more different sequences on saidsubstrate.
 19. A method of synthesizing a plurality of chemicalsequences, said chemical sequences comprising at least a first and asecond monomer, comprising the steps of: a) at a first region on asubstrate having at least a first and a second region, said first andsaid second region comprising a substrate protective group, activatingsaid first region to remove said substrate protective group in saidfirst region; b) exposing said first monomer to said substrate, saidfirst monomer further comprising a first monomer protective group, saidfirst monomer binding at said first region; c) activating said secondregion to remove said substrate protective group in said second region;d) exposing said second monomer to said substrate, said second monomerfurther comprising a second monomer protective group, said secondmonomer binding at said second region; e) activating said first regionto remove said first monomer protective group; f) exposing a thirdmonomer to said substrate, said third monomer binding at said firstregion to produce a first sequence; g) activating said second region toremove said second monomer protective group; and h) exposing a fourthmonomer to said substrate, said fourth monomer binding at said secondregion to produce a second sequence, said second sequence different fromsaid first sequence.
 20. A method of synthesizing a plurality ofchemical sequences, said chemical sequences comprising at least a firstand a second monomer, comprising the steps of: a) on a substrate havingat least a first and a second region deactivating said first region toprovide a first protective group in said first region; b) exposing saidfirst monomer to said substrate, said first monomer binding at saidsecond region; c) removing said protective group in said first region;d) deactivating said second region to provide a second protective groupin said second region; e) exposing said second monomer to saidsubstrate, said second monomer binding at said first region; f) removingsaid protective group in said second region; g) deactivating said firstregion to provide a protective group in said first region; h) exposing athird monomer to said substrate, said third monomer binding at saidsecond region to produce a first sequence; i) removing said protectivegroup in said first region; and j) exposing a fourth monomer to saidsubstrate, said fourth monomer binding at said first region to produce asecond sequence, said second sequence different than said firstsequence.
 21. A method of synthesizing at least a first polymer sequenceand a second polymer sequence on a substrate, said first polymersequence having a different monomer sequence from said second polymersequence, comprising the steps of: a) inserting a first mask betweensaid substrate and an energy source, said mask having first regions andsecond regions, said first regions permitting passage of energy fromsaid source, said second regions blocking energy from said source; b)directing energy from said source at said substrate, said energyremoving a protective group from first portions of said first polymerunder said first regions of said first mask; c) exposing a secondportion of said first polymer to said substrate to create a firstpolymer sequence; d) inserting a second mask between said substrate andsaid energy source, said second mask having first regions and secondregions; e) directing energy from said source at said substrate, saidenergy removing said protective group under said first regions of saidsecond mask from first portions of said second polymer; and f) exposinga second portion of said second polymer to said substrate, said secondportion of said second polymer binding with said first portion of saidsecond polymer to create a polymer 8 second sequence.
 22. The method asrecited in claim 21 wherein said energy is selected from the groupconsisting of ion beams, electron beams, gamma rays, x-rays,ultra-violet radiation, light, infra-red radiation, microwaves, electricfields, radiowaves, and combinations thereof.
 23. The method as recitedin claim 19 wherein said protective groups are photosensitive protectivegroups.
 24. The method as recited in claims 19 or 20 wherein said stepsof activating and deactivating are steps of applying light to selectedregions of said substrate.
 25. The method as recited in claims 19 or 20wherein said first and said second monomers are amino acids.
 26. Themethod as recited in claims 19, 20 or 21 further comprising a step ofscreening said first and said second sequences for affinity with a firstreceptor, said step of screening further comprising a step of exposingsaid substrate to said first receptor and testing for the presence ofsaid first receptor.
 27. The method as recited in claim 26 wherein saidstep of screening is a step of screening with antibodies.
 28. The methodas recited in claims 19, 20 or 21 wherein said substrate is selectedfrom the group consisting of a polymerized Langmuir Blodgett film,functionalized glass, germanium, silicon, polymers,(poly)tetrafluoroethylene, gallium arsenide, gallium phosphide, siliconoxide, silicon nitride and combinations thereof.
 29. The method asrecited in claim 19 wherein said protective group, said first monomerprotective group, and said second monomer protective group are selectedfrom the group consisting of ortho-nitrobenzyl derivatives,6-nitroveratryloxycarbonyl, 2-nitrobenzyloxycarbonyl, and mixturesthereof.
 30. The method as recited in claim 20 wherein said protectivegroup is a cinnamate group.
 31. The method as recited in claims 19 or 20wherein said first and second regions each have total areas of less than1 cm².
 32. The method as recited in claims 19 or 20 wherein said firstand second regions each have total areas of between about 1 μm² and10,000 μm².
 33. The method as recited in claim 24 wherein said light ismonochromatic coherent light.
 34. The method as recited in claim 19wherein said steps of activating are carried out with a solution incontact with said substrate.
 35. The method as recited in claim 34wherein said solution further comprises a monomer.
 36. The method asrecited in claim 26 wherein said receptor further comprises a markerselected from the group consisting of radioactive markers andfluorescent markers and wherein said step of testing for the presence ofthe receptor is a step of detecting said marker.
 37. The method asrecited in claims 19 or 20 wherein two of said first, said second, saidthird, and said fourth monomers are the same monomers.
 38. The method asrecited in claim 21 wherein the step of inserting a second mask is astep of translating said first mask from a first position to a secondposition.
 39. The method as recited in claim 21 wherein the step ofinserting a second mask is a step of rotating said first mask.
 40. Themethod as recited in claim 26 further comprising the step of exposingsaid substrate to a second, labeled receptor, said second, labeledreceptor binding at multiple sites on said first receptor.
 41. Themethod as recited in claim 40 wherein said first receptor is an antibodyof a first animal species and said second receptor is an antibodyderived from a second species and directed at said first species. 42.The method as recited in claim 19 wherein: a) said first monomerprotective group is removable upon exposure to a first wavelength oflight; b) said second monomer protective group is removable uponexposure to a second wavelength of light; c) said step of activatingsaid first region to remove said first monomer protective group is astep of exposing substantially all of said substrate to said firstwavelength of light; and d) said step of activating said second regionto remove said second monomer protective group is a step of exposingsubstantially all of said substrate to said second wavelength of light.43. A method as recited in claims 19 or 21 wherein said protectivegroups are of the form:

where R₁ is alkoxy, alkyl, halo, aryl, alkenyl, or hydrogen; R₂ isalkoxy, alkyl, halo, aryl, nitro, or hydrogen; R₃ is alkoxy, alkyl,halo, nitro, aryl, or hydrogen; R₄ is alkoxy, alkyl, hydrogen, aryl,halo, or nitro; and R₅ is alkyl, alkynyl, cyano, alkoxy, hydrogen, halo,aryl, or alkenyl.
 44. A method of screening a plurality of amino acidsequences for binding with a receptor comprising the steps of: a) on aglass plate having at least a first surface, said at least a firstsurface comprising a photoprotective material selected from the groupconsisting of nitroveratryloxy carbonyl and nitrobenzyloxy carbonyl,reacting said at least a first surface with t-butoxycarbonyl forstorage, said glass plate substantially transparent to at leastultraviolet light; b) exposing said at least a first surface to TFA toremove said t-butoxycarbonyl; c) placing said glass plate on a reactor,said reactor comprising a reactor space, said at least a first surfaceexposed to said reactor space; d) placing a mask at a first position onsaid glass plate, said mask comprising first locations and secondlocations, said first locations substantially transparent to at leastultraviolet light and said second locations substantially opaque to atleast ultraviolet light, said second locations comprising a lightblocking material on a first surface of said mask, said first surface ofsaid mask placed in contact with said glass plate; e) filling saidreactor space with a reaction solution; f) illuminating said mask withat least ultraviolet light, said ultraviolet light removing saidphotoprotective material from said at least a first surface of saidglass plate under said first locations of said mask; g) exposing saidfirst surface to a first amino acid, said first amino acid binding toregions of said at least a first surface from which said photoprotectivematerial was removed, said first amino acid comprising saidphotoprotective group at a terminus thereof; h) placing a mask incontact with said glass plate at a second position; i) illuminating saidmask with at least ultraviolet light, said ultraviolet light removingsaid photoprotective material from said at least a first surface of saidglass plate under said first locations of said mask; j) exposing said atleast a first surface to a second amino acid, said second amino acidbinding to regions of said at least a first surface from which saidphotoprotective material was removed, said second amino acid comprisingsaid photoprotective group at a terminus thereof; k) placing a mask incontact with said glass plate at a third position; l) illuminating saidmask with at least ultraviolet light, said ultraviolet light removingsaid photoprotective material from said at least a first surface of saidglass plate under said first locations of said mask; m) exposing said atleast a first surface to a third amino acid, said third amino acidbinding to regions of said at least a first surface from which saidphotoprotective material was removed; n) placing a mask in contact withsaid glass plate at a fourth position; o) illuminating said mask with atleast ultraviolet light, said ultraviolet light removing saidphotoprotective material from said at least a first surface of saidglass plate under said first locations of said mask; p) exposing said atleast a first surface to a fourth amino acid, said fourth amino acidbinding to regions of said at least a first surface from which saidphotoprotective material was removed, said at least a first surfacecomprising at least first, second, third, and fourth amino acidsequences; q) exposing said at least a first surface to an antibody ofinterest, said antibody of interest binding more strongly to at leastone of said first, said second, said third, or said fourth amino acidsequences; r) exposing said at least a first surface to a receptor, saidreceptor recognizing said antibody of interest and binding at multiplelocations thereof, said receptor comprising fluorescein; s) exposingsaid at least a first surface to light, said first surface fluorescingin at least a region where said more strongly bound amino acid sequenceis located; and t) detecting and recording fluoresced light intensity asa function of location across said at least a first surface.
 45. Amethod of identifying at least one peptide sequence for binding with areceptor comprising the steps of: a) on a substrate having a pluralityof polypeptides, each having a photoremovable protective group,irradiating first selected polypeptides to remove said protective group;b) contacting said polypeptides with a first amino acid to create afirst sequence, second polypeptides on said substrate comprising asecond sequence; and c) identifying which of said first or said secondsequence binds with said receptor.
 46. The method as recited in claim 45wherein said step of identifying further comprises a step of detectingthe presence of a marker selected from the group consisting ofradioactive markers and fluorescent markers in said receptor.
 47. Themethod as recited in claim 45 wherein said step of irradiating is a stepof masking a light source with a mask, said mask comprising firsttransparent regions and second opaque regions.
 48. The method as recitedin claim 47 wherein the step of identifying further comprises the stepsof: a) exposing a first receptor to said substrate; and b) exposing areceptor to said first receptor to said substrate, said receptor to saidfirst receptor comprising a marker.
 49. The method as recited in claim48 wherein said marker is selected from the group consisting ofradioactive markers and fluorescent markers.
 50. The method as recitedin claim 48 wherein said first receptor is an antibody from a firstspecies and said receptor to said first receptor is an antibody from asecond species directed at said first species.
 51. A method forscreening a plurality of polymers for biological activity comprisingexposing a receptor to a substrate having said plurality of saidpolymers on a surface thereof, each of said polymers occupying an areaof less than about 1 cm².
 52. A method for screening as recited in claim48 wherein said area is less than about 0.1 cm².
 53. A method as recitedin claim 48 wherein said area is less than about 10,000 μm².
 54. Amethod as recited in claim 48 wherein said area is less than about 100μm².
 55. Apparatus for preparation of a plurality of polymerscomprising: a) a substrate with a surface, said surface comprising areactive portion, said reactive portion activated upon exposure to anenergy source so as to react with a monomer; and b) means forselectively protecting and exposing portions of said surface from saidenergy source.
 56. Apparatus as recited in claim 55 wherein saidreactive portion further comprises a protective group, said protectivegroup of the form:

where R₁ is alkoxy, alkyl, halo, aryl, alkenyl, or hydrogen; R₂ isalkoxy, alkyl, halo, aryl, nitro, or hydrogen: R₃ is alkoxy, alkyl,halo, nitro, aryl, or hydrogen: R₄ is alkoxy, alkyl, hydrogen, aryl,halo, or nitro; and R₅ is alkyl, alkynyl, cyano, alkoxy, hydrogen, halo,aryl, or alkenyl.
 57. Apparatus as recited in claim 55 wherein saidreactive portion further comprises linker molecules.
 58. Apparatus asrecited in claim 57 wherein said linker molecules are selected from thegroup consisting of ethylene glycol oligomers, diamines, diacids, aminoacids, and combinations thereof.
 59. Apparatus as recited in claim 55wherein said means for selectively protecting further comprises a mask.60. Apparatus as recited in claim 55 wherein said means for selectivelyprotecting further comprises a light valve.
 61. Apparatus as recited inclaim 55 wherein said energy source is a light source.
 62. Apparatus asrecited in claim 55 wherein said reactive portion further comprises acomposition selected from the group consisting of nitroveratryloxycarbonyl, nitrobenzyloxy carbonyl, dimethyl-dimethoxybenzyloxy carbonyl,5-bromo-7-nitroindolinyl, hydroxy-2-methyl cinnamoyl, and 2-oxymethyleneanthraquinone.
 63. Apparatus for preparation of a substrate having aplurality of amino acid sequences thereon, said apparatus comprising: a)a substrate with a surface; b) a protective group on said surface, saidprotective group removable upon exposure to an energy source, saidenergy source selected from the group consisting of light, electronbeams, and x-ray radiation; c) means for directing said energy source atselected locations on said surface; and d) means for exposing aminoacids to said surface for binding to said surface.
 64. Apparatus forscreening polymers comprising a substrate with a surface, said surfacecomprising at least two predefined regions, said predefined regionscontaining different monomer sequences thereon, said predefined regionseach occupying an area of less than about 0.1 cm².
 65. Apparatus asrecited in claim 64 wherein said area is less than about 0.01 cm². 66.Apparatus as recited in claim 64 wherein said area is less than 10000μm².
 67. Apparatus as recited in claim 64 wherein said area is less thanabout 100 μm².
 68. Apparatus as recited in claims 64, 65, 66, or 67wherein said monomer sequences are substantially pure within saidpredefined regions.
 69. A substrate for screening for biologicalactivity, said substrate comprising 10³ or more different ligands on asurface thereof in predefined regions.
 70. A substrate as recited inclaim 69 wherein said substrate comprises 10⁴ or more different ligandsin predefined regions.
 71. A substrate as recited in claim 69 whereinsaid substrate comprises 10⁵ or more different ligands in predefinedregions.
 72. A substrate as recited in claim 69 wherein said substratecomprises 10⁶ or more different ligands in predefined regions.
 73. Asubstrate as recited in claims 69, 70, 71, or 72 wherein the ligands arepeptides.
 74. A substrate as recited in claim 64 wherein said ligandsare substantially pure within said predefined regions.
 75. Apparatus forscreening for biological activity comprising: a) a substrate comprisinga plurality of polymer sequences, said polymer sequences attached to asurface of said substrate at known locations on said substrate, each ofsaid sequences occupying an area of less than about 0.1 cm²; b) meansfor exposing said substrate to a receptor, said receptor marked with afluorescent marker, said receptor binding with at least one of saidsequences; and c) means for detecting a location of said fluorescentmarker on said substrate.
 76. Apparatus for forming a plurality ofpolymer sequences comprising: a) a substrate, said substrate having atleast a first surface and a second surface, said second surfacecomprising a photoremovable protective material, said substratesubstantially transparent to at least light of a first wavelength; b) areactor body, said reactor body having a mounting surface with areaction fluid cavity therein, said second surface maintained in asealed relationship with said mounting surface; and c) a light sourcefor producing light of at least said first wavelength and directed at asurface of said substrate.
 77. Apparatus as recited in claim 76 whereinsaid light source is directed at said first surface.
 78. Apparatus asrecited in claim 76 further comprising a mask, said mask placed betweensaid light source and said first surface, said mask having first regionssubstantially transparent to said first wavelength of light and secondregions substantially opaque to said first wavelength of light. 79.Apparatus as recited in claim 76 wherein said cavity comprises a fluidinlet and a fluid outlet, said fluid inlet connected to a pump forflowing reaction fluids through said cavity.
 80. Apparatus as recited inclaim 76 wherein said cavity further comprises a plurality of raisedsections.
 81. Apparatus as recited in claim 78 wherein said mask furthercomprises a glass plate.
 82. Apparatus as recited in claim 81 whereinsaid opaque regions on said mask comprise chrome.
 83. Apparatus asrecited in claim 76 wherein at least a portion of said second surfacecomprises a second photoremovable protective group, said secondphotoremovable protective group activatable upon exposure to light of asecond wavelength.
 84. Apparatus as recited in claim 76 furthercomprising first and second gaskets on said mounting surface and meansfor maintaining a vacuum between said first and second gaskets. 85.Apparatus as recited in claim 76 wherein said substrate has a thicknessof less than 1 mm.
 86. Apparatus as recited in claim 76 wherein saidsubstrate has a thickness of less than 0.5 mm.
 87. Apparatus as recitedin claim 76 wherein said substrate has a thickness of less than 0.05 mm.88. Apparatus as recited in claim 78 wherein said mask is in directcontact with said substrate.
 89. Apparatus as recited in claim 88wherein opaque regions of said mask are placed in direct contact withsaid substrate.
 90. Apparatus as recited in claim 76 further comprisinga liquid crystal light valve for selectively controlling exposure oflight to said substrate.
 91. Apparatus as recited in claim 76 furthercomprising a fiber optic faceplate between said light source and saidsubstrate.
 92. Apparatus as recited in claim 76 further comprising amolecular microcrystal between said light source and said substrate. 93.Apparatus as recited in claim 76 wherein said cavity comprises lightabsorptive materials.
 94. Apparatus as recited in claim 93 wherein saidlight absorptive material is N,N-diethylamino 2,4-dinitrobenzene. 95.Apparatus as recited in claim 76 wherein said cavity is filled with acarrier solution.
 96. Apparatus as recited in claim 95 wherein saidcarrier material comprises a material selected from the group of1-hydroxybenzotriazole, dimethylformamide, diisopropylethylamine, andbenzotriazolyl-n-oxy-tris(dimethylamino)phosphoriumhexafluorophosphate.97. Apparatus as recited in claim 76 wherein said substrate is a fiberoptic faceplate.
 98. Apparatus for detection of fluorescent markedregions on a substrate comprising: a) a light source for directing lightat a surface of said substrate; b) a means for detecting lightfluoresced from said surface in response to said light source; c) meansfor translating said substrate from a first position to a secondposition; and d) means for storing fluoresced light intensity as afunction of location on said substrate, said means for storing connectedto said means for translating and said means for detecting. 99.Apparatus as recited in claim 98 further comprising video display meansfor displaying light intensity as a function of location on saidsubstrate.
 100. Apparatus as recited in claim 98 wherein said means fordetecting comprises a photomultiplier tube and a photon counter. 101.Apparatus as recited in claim 99 wherein said means for directing lightfurther comprises a dichroic mirror, said mirror reflecting light at awavelength of said light source and passing said fluoresced light. 102.Apparatus as recited in claim 100 wherein said light source is a laserlight source.
 103. Apparatus as recited in claim 101 wherein said meansfor storing is a programmed digital computer.
 104. Apparatus as recitedin claim 102 further comprising a microscope, said light source directedat said substrate through said microscope, said means for detectingreceiving light from said microscope.